HK1168544A - Human protein scaffold with controlled serum pharmacokinetics - Google Patents

Description

Human protein scaffolds with controlled serum pharmacokinetics

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application serial No. 61/167844, filed on 8/4/2009, the contents of which are incorporated herein by reference in their entirety for all purposes.

Statement of rights to invention under federally sponsored research and development

The invention was made with the funding of fund number CA086306 awarded by the National Institutes of Health with government support. The government has certain rights in the invention.

Sequence listing, form or computer program listing attachment submitted on optical disc

Is free of

Technical Field

The present invention relates to constructs, compositions thereof, and uses thereof, wherein the constructs comprise human serum albumin domain III as a scaffold having one or more targeting moieties and one or more imaging, diagnostic or therapeutic moieties bound thereto.

Background

Over the last decade, combinatorial library technology has produced a large number of molecules, including peptides, aptamers, and small chemical molecules, that can be selected to bind to a target or tissue with high specificity and affinity (Aina et al, 2007; Barbas and White, 2009; Bembenek et al, 2009). However, when administered in vivo, these molecules tend to exhibit suboptimal Pharmacokinetics (PK) characterized by transient serum persistence and failure to accumulate at the target site to sufficient levels for imaging or therapeutic applications. To address this problem, these targeting molecules may be bound to a scaffold. Various scaffolds have been described in recent reviews (Gronwall and Stahl, 2009; Nuttall and Walsh, 2008). However, they are either of non-human origin (e.g., affibody (affibody), derived from Staphylococcal (staphylococcus) protein a (Friedman et al, 2007), camelid and shark single domain antibody isoforms (Saerens et al, 2008), cystine-knot small proteins derived from plant cyclic peptides (simons et al, 2008)), or do not provide controlled PKs (ankyrin, mimobulin (adnectin), avimer, lipocalin and anticalin (Nuttall and Walsh, 2008)).

Human serum albumin (HSA; 67kDa) is the most abundant protein in humans (30-50g/L) and has been incorporated into approved pharmaceuticals (i.e., Albuferon by Novartis Inc. (Novartis)). In preclinical studies, HSA has been successfully used as a carrier molecule for drug delivery (Burger et al, 2001; Kratz et al, 2000; Wosikowski et al, 2003) and as a vector for gene delivery (Aina et al, 2007). As fusion proteins, HSA has been shown to improve the PK of molecules, such as interferon- α (Osborn et al, 2002), interleukin-2 (Melder et al, 2005), recombinant bispecific antibody molecules (Muller et al, 2007) or scFv antibody fragments (Yazaki et al, 2008). Like IgG, HSA interacts with the neonatal Fc receptor-FcRn, also known as the Brambell receptor (Chaudhury et al, 2003). This interaction is responsible for prolonging the serum persistence of albumin. Briefly, albumin molecules are taken from the circulation into the endosomes of vascular endothelial cells by fluid phase endocytosis. In the primary endosome (-pH 6.5), albumin binds to the FcRn located within this component. After the endosome fuses with the lysosome, unbound components in the endosome are released for degradation, while FcRn-bound albumin is protected. Endosomes circulate back to the apical surface of endothelial cells, towards the neutral environment of circulation (pH 7.4), where albumin is released back into the blood by FcRn. In particular, HSA domain III (DIII; 23kDa) has been shown to bind to FcRn in a pH-dependent manner (Chaudhury et al, 2006). The three conserved histidine residues in HSA DIII (H535, H510, and H464) were postulated to play a role in the HSA-FcRn interaction (Bos et al, 1989; Chaudhury et al, 2006).

Currently, the most successful targeting agents for cancer therapy in the clinic are intact antibodies (e.g., trastuzumab, rituximab, bevacizumab). The advantages of using antibodies, in addition to their superior target affinity and specificity and good safety, are that they also possess the Pharmacokinetics (PK) necessary to achieve a therapeutic effect. The long-term circulating persistence of antibodies is mainly due to the interaction of their Fc domain with FcRn. The Fc domain of an antibody is twice the size of HSA DIII, and in addition, the Fc domain interacts with other endogenous Fc receptors. This biological function can cause adverse side effects in clinical applications. The disadvantages of antibodies also include certain limitations of target accessibility, but primarily their production process is time consuming and highly laborious, which also increases the cost of antibody pharmaceuticals.

Other targeting moieties, including peptides and aptamers, can also be selected to exhibit nanomolar affinities and high specificity for different targets, and are much faster and less expensive to produce than antibodies. The major disadvantage is that these low molecular weight targeting agents are usually cleared from the circulation very rapidly, with a typical serum half-life of several minutes. This leads to lower target uptake and limits their potential for clinical applications in diagnostic imaging and therapy. The ability to modulate within the ideal PK of targeted imaging and therapeutic agents is of great value in modern medicine. Compositions and methods for in vivo therapy and imaging using molecules with diverse circulating half-lives that target biomolecules and prolong serum persistence without significant changes in molecular weight are provided. The present invention addresses the need for low molecular weight, low or non-immunogenic agents that provide tumor targeting molecules, such as peptides, aptamers or small chemicals, with the appropriate pharmacokinetic properties required for in vivo applications, including imaging and/or therapy.

Summary of The Invention

In a first aspect, the invention provides the use of HSA DIII as a scaffold in the preparation of a construct comprising HSA DIII and one or more small molecule targeting agents coupled to HSA DIII, and one or more imaging or therapeutic moieties coupled to HSA DIII. The HSA-DIII scaffold or carrier can be modified to provide constructs (PK) with specific pharmacokinetics and to provide the potential for multivalency and/or multispecific and functional group-binding residues.

In some embodiments, the constructs provided herein comprise a) a protein scaffold, wherein the scaffold comprises domain III, domain IIIa, or domain IIIb of human serum albumin, or a variant selected for altered FcRn receptor binding properties thereof; b) a targeting moiety covalently linked to the protein scaffold; and c) a therapeutic moiety or an imaging moiety covalently linked to the protein scaffold.

In another aspect, the invention provides a method of detecting a biomolecule associated with a disease or condition in a subject by administering a construct of the invention to a subject suspected of having or suffering from the disease or condition, wherein a targeting moiety of the construct binds to the biomolecule and the imaging agent to which the construct binds is measured. In some embodiments, the presence or absence of the disease or disorder is diagnosed.

In another aspect, the invention provides a method of targeted treatment of a disease or condition associated with the presence of overexpression of a biomolecule in a tissue, the method comprising administering to a subject having the disease or condition a therapeutically effective amount of a construct according to the invention, wherein the targeting moiety of the construct binds to the biomolecule and the therapeutic agent of the construct treats the disease or condition in the tissue or cell associated with the presence of the biomolecule.

In another aspect, the present invention provides a library of modified domain III proteins having different target specificities and predetermined FcRn affinities for use as scaffolds in the design of the targeted imaging and therapy constructs of the invention. In another embodiment, the invention provides nucleic acids encoding one or more domain III scaffolds and variants thereof for use according to the invention. In still further embodiments, the invention provides vectors comprising a nucleic acid operably linked to a gene regulatory factor to control domain III scaffold expression, and cells containing the vector or nucleic acid.

In all embodiments and aspects of the invention, some embodiments provide that the construct does not contain one or both of domain I or domain II of HSA, or the construct does not contain a sequence of more than 5, 10, 15, or 20 consecutive amino acids of domain II of HSA.

Brief description of the drawings

FIG. 1 (A) Gene Assembly of construct Db-DIII. A leader signal peptide secreted by the L-mammalian cell; variable region of light chain of antibody (V)_L) And heavy chain variable region (V)_H) Single chain variable fragments (scFv, 25kDa) were formed by 8 amino acid (glycine, serine rich) linker ligation. The scFv was linked to the HSA DIII gene by an 18 amino acid linker. DIII is flanked by SpeI and EcoRI restriction sites within the cassette to facilitate the exchange of one DIII with another DIII (e.g., WT exchange is H535A, etc.). (B) Cartoon representation of the Db-DIII protein, wherein two scFv-DIII molecules form a non-covalent dimer.

FIG. 2 (A) SDS-PAGE of 4 Db-DIII proteins. Lanes 1, 2 and 3 are H535A, H510A and H464A under NR condition, respectively, and lane 5 is WT under R condition. (B) Western blot of Db-DIIIWT under NR (lane 2; detection with AP-conjugated anti-mouse Fab mAb) and R (lane 3; detection with HRP-protein L). (C) Size exclusion chromatography using a Superdex 200 column and a flow rate of 0.5 ml/min. The Db-DIII WT protein eluted at 28.17 minutes. The resulting purity was estimated to be about 98% by peak integration.

FIG. 3 (A) PyMOL model of HSA DIII consisting of hemi-domains of DIIIa (green) and DIIIb (yellow). Six disulfide bridges are shown in red. Arrows mark the positions of residues H535, H510 and H464. The H464 residue located in DIIIa was mutated to a to produce DIII H464A variant. Amino acids H535 and H510 located at DIIIb were each exchanged with a to produce DIII H535A and DIII H510A variants. (B) Docking models for HSA DIII (green) and FcRn (orange) to FcRn (orange) molecules. Red is the residue on FcRn involved in IgG binding and residues interacting with each other at the interface of FcRn and DIII molecules are shown in blue. (C) Model of bivalent Db-DIII molecules, where each Db carries two DIII proteins at the C-terminus. The scFv monomers that make up Db are light green and dark green, the 18 amino acid linker is blue, and the DIII molecule is yellow.

FIG. 4. mouse PET/CT imaging of athymic nude mice xenografted with CEA-positive LS174T (left) and CEA-negative C6 (right) tumors. The mice are injected with¹²⁴I-labeled Db-DIII protein (WT, H535A, H510A, or H464A) and anti-CEA Db as reference. Mice were imaged for 10 min at 5 different time points, showing coronal sections. The mutually registered images, including PET/CT, are used for anatomical reference of tumor and organ positions.

Fig. 5 (a) tumor versus soft tissue ROI analysis of PET images. (B) The resulting blood activity curves were quantified by radioactivity (% ID/g) of PET images at each time point.

Figure 6 cell binding identification HSA, DIII WT, H535A, H510A and H464A proteins coupled with Alexa 647 were incubated at elevated concentrations with 293 cells transduced with human FcRn. Alexa-coupled HSA was incubated with untransfected 293 cells as a control.

FIG. 7 Balb/c mice¹³¹Blood activity profiles of labeled HSA and DIII proteins.

FIG. 8 DNA and translated protein sequences of Db-DIII. Specific sequences and start sites for the following DNA and protein segments are indicated: restriction enzyme digestion site, Kozac sequence, leader-secretory signal peptide, V_L8 interdomain peptide linker of amino acids, V_HA linker of 18 amino acids between Db and HSA DIII; histidine residues H535, H510A, and H464, which were independently mutated to alanine to yield the Db-DIII variant, and two stop codons followed by restriction sites.

Figure 9. DNA and translated protein sequence of a. diii WT. The important sequences and start sites for the following DNA and protein segments are shown: restriction enzyme digestion sites used in cloning, Kozac sequence, leader sequence, start of HSA DIIIa, HSA DIIIb, histidine residues H535, H510 and H464, c-Myc peptide, followed by two stop codons for restriction enzyme digestion sites; HSA DIIIa; and c.hsa DIIIb.

Fig. 10.¹²⁴I Small animal PET/CT imaging of the CEA peptide-DIIIb conjugate. Static scans were performed at 10 min 4, 20 and 27 hours post injection, showing coronal sections. Arrows mark CEA positive (LS174T) and CEA negative (C6) tumors.

Detailed Description

The invention provides for the use of HSA DIII as a scaffold in the preparation of a construct comprising HSA DIII and one or more small molecule targeting agents coupled to HSA DIII, and one or more imaging or therapeutic moieties coupled to HSA DIII. The HSA-DIII scaffold or carrier can be modified to provide constructs with specific Pharmacokinetics (PK) by selecting amino acid sequence modifications within domain III that affect FcRn receptor binding or provide complementary sites to bind targeting, therapeutic and imaging moieties, and provide the potential for multivalent and/or multispecific binding as well as residues of binding functionalities.

We have found that low molecular weight tumor targeting molecules bound to, grafted to or displayed on HSA domain III protein scaffolds characterized by intrinsic serum stability, can subsequently achieve improved pharmacokinetic properties and target uptake. Maximized tumor accumulation can translate into a stronger signal in imaging applications or adequate drug load delivery in therapy. In addition, the HSA DIII scaffold can provide residues to couple functional groups (e.g., radionuclides, cytotoxic drugs, toxins), and can also enhance the solubility of hydrophobic targeting molecules. The scaffold is advantageous because it can 1. be substantially non-immunogenic, 2. provide optimal serum persistence (tunable) for different applications, 3. low molecular weight, promote extravasation, tumor penetration and renal clearance (< 60kDa), which is preferred for imaging applications, 4. a platform to increase the functional affinity of targeting molecules by using affinity effects (2-3 targeting molecules on the same scaffold) or introducing multiple specificities, and 5. soluble in serum, making its surface bound molecules also soluble.

In some embodiments, the complexes/constructs provided herein comprise a) a protein scaffold, wherein the scaffold comprises domain III, domain IIIa, or domain IIIb of human serum albumin, or a variant thereof; b) a targeting moiety covalently linked to the protein scaffold; and c) a therapeutic moiety or an imaging moiety covalently linked to the protein scaffold. The targeting moiety is a ligand that binds to a receptor of a target tissue or cell. Thus, in some embodiments, the targeting moiety is an antibody, or more preferably an immunologically active fragment thereof, that is capable of binding a biomolecule (e.g., a tumor-specific antigen) of a target tissue or cell. The antibody is preferably a scFv diabody, triabody or minibody. In some embodiments, the targeting moiety is a nucleic acid aptamer. The targeting moiety is capable of binding a biomolecule present in the subject or on a target tissue or cell of the subject. The biomolecule is preferably a tumor specific antigen or other biomolecule whose presence in a target tissue or cell is associated with or overexpressed in a disease or health condition. Proposed tumor-specific antigens include, but are not limited to, CEA, CD20, HER2/neu, PSCA, PSMA, CA-125, CA-19-9, c-Met, MUC1, RCAS1, Ep-CAM, Melan-A/MART1, RHA-MM, VEGF, EGFR, integrin, and fibronectin ED-B. Thus, in some embodiments, the target tissue or cell is a cancerous tissue or cell.

In some embodiments of the invention, at least one or all of the targeting moiety, imaging moiety or therapeutic moiety is covalently bound to the scaffold via a non-peptide linker or a non-peptide bond. In other embodiments, at least one or all of the targeting moiety, imaging moiety, or therapeutic moiety is covalently bound to the scaffold by a heterobifunctional crosslinker, a homobifunctional crosslinker, a zero-length crosslinker, a disulfide bond, or a physiologically cleavable crosslinker. The linker of the targeting, imaging and therapeutic moieties is preferably 2-50 atoms in length (e.g., 2-10, 4-40, 10-30 atoms in length). The domain III scaffold may bind more than one targeting, imaging or therapeutic moiety.

In some embodiments, a small peptide or other targeting moiety (aptamer, chemical) is genetically fused or coupled to HSA DIII; in other embodiments, a protein (e.g., an antibody, antibody fragment, enzyme, receptor ligand, cytokine, chemokine, growth factor) is fused to the HSA DIII scaffold as a targeting moiety; or 3) nanoparticles, diamagnetic materials, quantum dots, radionuclides, or compounds can be combined with the HSADIII scaffold as an imaging moiety.

For molecular imaging purposes, various radionuclides can be conjugated to a protein scaffold for detection using gamma or SPECT cameras or PET scanners. Diamagnetic materials can be coupled for MR imaging. For optical imaging, the HSA DIII scaffold can be fused with a fluorescent dye, protein, or bioluminescent enzyme (e.g., firefly, renilla, or gaussian (Gaussia) luciferase).

For therapeutic applications, therapeutic radionuclides, cytotoxic drugs, toxins, cytokines, enzymes, or other therapeutic moieties may be combined with the targeted HSA DIII scaffold for target-specific delivery to tumors. In some embodiments, the linkage to DIII is susceptible to cleavage under physiological conditions (e.g., enzymatic cleavage, such as acidic cleavage within a lysosome).

The present invention provides the advantage of providing a low or non-immunogenic human HSA domain III protein (e.g., 23 or 11kDa) of lower molecular weight than HSA, which has the ability to alter or prolong the serum persistence of the bound molecule to a defined extent.

In any of the above embodiments, HSA domain III is preferably wild-type and has a mutation at H535, H510 or H464 to alter binding of said domain to the FcRn receptor. In some embodiments, the mutation is H535A, H510A, H464A; H535A and H510A and H464A; H535A and H464A; H535A and H510A; or H510A and H464A. In some of the above embodiments, the protein scaffold consists essentially of HSA domain III, domain IIIa, or domain IIIb, or a polypeptide that is substantially identical in sequence to these three domains.

In some embodiments, the therapeutic moiety of the construct is a drug. For example, the therapeutic moiety may be a therapeutic radionuclide, cytotoxic drug, cytokine, chemotherapeutic agent, radiosensitizer, or enzyme. In further embodiments, a plurality of therapeutic moieties are covalently linked to the protein scaffold.

In other embodiments, the construct comprises an imaging agent. Suitable imaging agents include, but are not limited to, radionuclides, diamagnetic materials, paramagnetic particles, fluorophores, chromogenic agents, quantum dots, nanoparticles, and bioluminescent enzymes. The scaffold may be covalently bound to one or more imaging agents.

In some embodiments, the construct is monovalent or multivalent. For example, the targeting moiety or other component of the construct (e.g., a targeting moiety bound to a DIII scaffold, see fig. 3) may itself be monovalent, bivalent, trivalent, or multivalent, each component being covalently linked or bound to its HSA DIII scaffold.

In another aspect, the invention provides a method of detecting a biomolecule associated with a disease or condition in a subject by administering a construct of the invention to a subject suspected of having or suffering from the disease or condition, wherein a targeting moiety of the construct binds to the biomolecule and detecting the imaging agent to which the construct binds. In some embodiments, the presence or absence of the disease or disorder is diagnosed based on the detecting. For example, where the biomolecule is a tumor-specific antigen that is overexpressed in a cancer, the presence or absence of the cancer associated with the tumor-specific antigen can be determined by administering the construct of the invention to a subject and detecting the imaging moiety bound to the construct in the subject. The location of the imaged portion of the construct at the tumor site measured indicates the presence of cancer. In some embodiments, the serum persistence of the construct or imaging agent is fine-tuned by selecting a domain III polypeptide containing a mutation that alters the affinity of domain III (diii) for the FcRn receptor. Radionuclides for imaging include, but are not limited to, I-131, I-123, In-111, and Tc-99m for SPECT imaging and F-18, I-124, Cu-64, Y-86 for PET imaging.

In another aspect, the invention provides a method of targeted treatment of a disease or condition associated with the presence of overexpression of a biomolecule in a tissue, the method comprising administering to a subject having the disease or condition a therapeutically effective amount of a construct of the invention, wherein the targeting moiety of the construct binds to the biomolecule and the therapeutic agent of the construct treats the disease or condition in the tissue or cells associated with the presence of the biomolecule. For example, in some embodiments, the targeting moiety binds to a tumor-specific antigen of a cancer, the disease or disorder to be treated is the cancer, and the therapeutic agent is a therapeutic radionuclide, cytotoxic drug, cytokine, or chemotherapeutic agent. Therapeutic chemotherapeutic agents that can bind to targeted DIII include, but are not limited to: gemcitabine, doxorubicin, vincristine, topotecan, irinotecan. Examples of toxins that can be coupled to DIII are auristatin or pseudomonas exotoxin a. Therapeutic radionuclides include, but are not limited to: beta emitters-Y-90, Lu-177, I-131, Sm-153 and Sr-89; and alpha emitters-Ra-223, Th-227, Ac-225, At-211, Bi-212, and Bi-213. The DIII scaffold can be covalently bound to one or more therapeutic agents.

In some embodiments, the therapeutic and imaging functionalities can be bound to the same target-specific DIII platform simultaneously for the following applications: the drug conjugates are observed to target to the tumor/disease site, the course of therapy is monitored by molecular imaging and the pathway of metabolic clearance is determined.

Accordingly, the present invention also provides 1) a pharmaceutical or diagnostic composition comprising the above-described therapeutic and imaging construct and a physiologically acceptable excipient or carrier; 2) the use of a therapeutic construct according to the invention in the manufacture of a medicament for the treatment of a disease or condition; and the use of the imaging constructs of the invention in the manufacture of a diagnostic for the detection of a disease or condition.

The present invention proposes chemical coupling of tumor targeting peptides to selected DIII platforms. For example, available¹²⁴I(t^1/24.2 days) or⁶⁴Cu(t_1/212.7 hours) labeled proteins were injected into tumor-bearing subjects and the targeting of these proteins to antigen-positive tumors was assessed by PET imaging. Expression of these variable region sequences on the natural antibody backbone, or as scFv, triabody, diabody or minibody, labeled with a radionuclide, is particularly useful in vivo detection of target-bearing cells. Such expression on the backbone or natural antibody backbone is preferably used not only to target but also to block the function of target biomolecules and/or to kill or inhibit the growth or proliferation of cells carrying these molecules in vivo.

In another aspect, the invention provides a library of modified domain III proteins having different predetermined FcRn affinities for use as scaffolds in the design of the targeted imaging and therapy constructs of the invention. In another embodiment, the invention provides nucleic acids encoding one or more domain III scaffolds and variants thereof for use according to the invention. In still further embodiments, the invention provides vectors comprising a nucleic acid operably linked to a gene regulatory factor to control domain III scaffold expression, and cells containing the vector or nucleic acid.

The three conserved histidine residues in HSA DIII-H535, H510, and H464 are hypothesized to play a role in HSA-FcRn binding, and variants at these residues are specifically proposed. To assess the ability to modulate the HSA-FcRn interaction, we generated and expressed a fusion protein consisting of an anti-CEA antibody (Db, a non-covalent dimer of two scfvs; 55kDa) and wild-type HSA DIII (WT, no mutation) or one of 3 variants thereof, each incorporating one mutation of H535, H510 or H464 to an alanine residue. By using¹²⁴I-labeled Db-DIII protein injection Small animal PET/CT imaging of xenograft athymic mice showed that HSA DIII can prolong serum persistence of Db while maintaining tumor targeting. Image analysis and biodistribution studies showed that Db-DIII WT lasted the longest in the cycle, with an estimated Mean Residence Time (MRT) of 56.7 hours, followed by Db-DIII H535A (25 hours) > H510A (20 hours) > H464A (17 hours) and Db (2.9 hours). Generation HSA DIIIWT and variants (H535A, H51)0A and H464A) and the DIIIa subdomain (amino acid residues 384-492; 14.2kDa) and the DIIIb subdomain (510-); 12.2 kDa). By intravenous injection of each in Balb/c mice¹³¹The I-labeled DIII proteins evaluated their pharmacokinetic properties in blood. Blood was taken from the tail at 8 different time points (0-72 hours) and radioactivity was counted in a gamma well counter. Terminal serum half-life (t) of each protein_1/2β) was determined as follows: DIII WT (15.3 hr), H535A (10.7 hr), H464A (10.2 hr), H510A (9.75 hr), DIIIa (8.93 hr) and DIIIb (6.87 hr) compared to intact HSA protein (17.3 hr). The DIII protein is selected to be used as a scaffold for grafting or chemically coupling tumor targeting molecules (peptides, aptamers, or small chemical moieties) and directly for generating combinatorial display libraries. Target-specific scaffolds with pharmacokinetics suitable for diagnostic use may be used for imaging applications. Alternatively, potential antineoplastic drugs can be conjugated to targeting scaffolds with optimal characteristics for therapeutic use and for cancer therapy.

The present invention also provides a docking model that shows that two more residues in DIII are important for the interaction with FcRn (i.e., glutamate residues E505 and E531). Thus, some embodiments of the invention provide variants and nucleic acids of DIII, DIIIa or DIIIb protein scaffolds, and vectors and transduced cells containing the same, which variants have amino acid substitutions at E505 and/or E531, and otherwise are substantially identical or identical to the sequence of domain III, IIIa or IIIb of HSA. In some embodiments, one or both of these residues is substituted with aspartic acid, in other embodiments, one or both of these residues is substituted with an uncharged amino acid, and in still further embodiments, one or both of E505 and E531 is substituted with alanine or glycine. In other embodiments, the substitutions are E505D, A, G, I, V or L or E531D A, G, I, V or L substitutions that interfere with DIII binding to FcRn and thereby modulate the circulating half-life of the target-specific DIII imaging or therapeutic agent.

The targeting moiety can be any molecule that is capable of binding to the target biomolecule. In some embodiments, the target molecule is extracellularA tumor specific antigen present on the surface. The targeting moiety may be an antibody, or more preferably an antibody fragment having affinity for a molecule recognized by said antibody. In a preferred embodiment, the antibody is a scFv, diabody, minibody or triabody. In some embodiments, the targeting moiety is a nucleic acid aptamer or a small peptide (e.g., 5-30 amino acids, 2-20 amino acids in length) that binds to the biomolecule. The targeting moiety preferably has a high affinity for the biomolecule, K_dLess than 100nM, 30nM, 10nM or 1 nM. Furthermore, the use of multiple (2, 3,4 or more) such or lower affinity targeting moieties per scaffold for a target biomolecule may facilitate binding to the target cell through affinity effects.

The term "imaging agent or moiety" as used herein refers to an agent or moiety that is capable of providing a detectable signal, either directly or through interaction with a complementary member of a signal producing system. Preferably, the construct generates a signal in the subject, which signal is externally detectable.

"therapeutic moiety" refers to an agent that can be used to treat a disease or disorder or have some other desired benefit to a target tissue and/or cell of a subject. The therapeutic moiety may be a therapeutic drug, hormone, cytokine, interferon, antibody or antibody fragment, aptamer, enzyme, polypeptide, toxin, cytotoxin, or chemotherapeutic agent. The therapeutic moiety may be a radiosensitizer.

The linker used to attach the targeting, imaging or therapeutic moiety to the scaffold may comprise a covalent bond or a chain length of 1-100 atoms or longer. The linker may comprise carbon, nitrogen, sulfur, or oxygen atoms in the chain. Carbon chains (e.g., from about 5 to about 50 carbons) are specifically contemplated. The linker may comprise a nucleic acid or an amino acid. Examples of carbon chains as linkers include, but are not limited to, alkyl, alkene, or aldehyde. The carbon chain may be substituted, unsubstituted, unbranched or branched with one or more substituents. The linker may comprise a length of about 5 nanometers to about 50 nanometers, 3 to 30nm, and more preferably about 5 to 10 nm. Examples of linkers may include, but are not limited to, carbon chains of about 10 to about 20 carbons in length. Polyalkylene glycol (e.g., PEG) linkers are also contemplated. The linker may comprise a non-peptide bond. Linkers include, but are not limited to, heterobifunctional, homobifunctional, zero-length, disulfide, or physiologically cleavable crosslinkers. The linker of the targeting, imaging and therapeutic moieties is preferably 2-80 atoms in length (e.g., 2-10, 4-40, 10-30, 2-50 atoms in length). When the fused agent is a polypeptide, fusion proteins of domain III and at least one targeting, imaging or therapeutic agent are also contemplated. It is also contemplated that the targeting, imaging, and therapeutic agents may not be each linked to the scaffold as a fusion protein, or linked to the scaffold via another amino acid or via a peptide bond.

The imaging agent and therapeutic moiety can be directly coupled to the DIII protein scaffold using conventional methods well known in the art. Radioactive and non-radioactive labels are routinely used (for a review of enzymatically, photochemically and chemically labeled nucleic acids and proteins see Bioconjugate Techniques (Bioconjugate technology), 2 nd edition, Greg t. hermanson, Academic Press, Inc., 2008, p. 1202).

Aptamers are oligonucleotide molecules that bind to a specific target molecule. Aptamers are usually generated by selection operations in large random sequence libraries. Nucleic acid aptamers can be obtained by repeated rounds of in vitro selection or equivalent SELEX (systematic evolution of exponentially enriched ligands) to bind to different molecular targets such as small molecules, proteins, nucleic acids, or even cells and tissues using methods well known in the art. As is well known in the art, combinatorial libraries based on complex nucleic acids (e.g., > 10 per library) can be generated¹⁴Seed nucleic acids) using a process known as SELEX (see U.S. patent publication No. 20090004667, incorporated herein by reference). SELEX is an iterative process of incubating a library of pools of random RNA sequences with a selected protein target. The interacting RNA is then separated from unbound RNA and amplified by reverse transcription followed by polymerase chain reaction amplification (RT/PCR). The enriched RNA pool can be generated by in vitro transcription using a DNA template with a mutant T7 RNA polymerase that allows for the incorporation of a 2' fluoro-modified pyrimidine. These modifications make the RNA more nuclease resistant. The step that results in the enriched RNA pool is called a "screening round". Targeting protein targetsThe screening rounds of (a) are generally continued until the progression of binding affinity reaches a plateau. Individual clones can then be isolated from the pool and sequenced. Aptamers can provide molecular recognition properties comparable to or even superior to antibodies. In addition to specific recognition, aptamers offer advantages over antibodies. Aptamers can be fully engineered in vitro and can be conveniently made by chemical synthesis. Aptamers also have desirable storage and dissolution properties and cause little or no immunogenicity in therapeutic applications. Aptamers useful in the invention may be nucleic acids that bind with high affinity (e.g., Kd of less than 100nM, 10nM or 1nM) to the ED-B of CEA, CD20, HER2/neu, PSCA, PSMA, CA-125, CA-19-9, c-Met, MUC1, RCAS1, Ep-CAM, Melan-A/MART1, RHA-MM, VEGF, EGFR, integrins and fibronectin. Aptamers are preferably 10-30, 10-20 or 15-25 nucleic acids in length.

The amino acid sequence of domain III according to the invention is that of the HSA domain III, IIIa or IIIb (see FIGS. 9a, b, c, respectively) or is substantially identical thereto. Domain III 1) comprises, consists of or consists essentially of the amino acid sequence of: preferably, the polypeptide has greater than about 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over its entire sequence or over a stretch of at least about 15, 20, 25, 50, 75, 100, 125, 150 or more amino acids and is capable of binding to the fcrn (brambell) receptor as compared to the polypeptide shown in figure 9a, 9b or 9 c. Domain III (amino acid residue 384-585) has two subdomains-DIIIa (amino acid residue 384-492) and DIIIb (amino acid residue 510-585). (see Sugio et al, protein engineering, Vol.12, 6, 439-446, 1999, 6.Y., HSA sequence and structure, incorporated herein by reference). In some embodiments, the domain III of the claims is a polypeptide comprising, consisting of, or consisting essentially of HSA domain III, domain IIIa, or domain IIIb disclosed herein and H535, H510, or H464 variants thereof.

Domain III, domain IIIa or domain IIIb according to the invention may be conservatively modified variants of the polypeptides shown in figures 9a, b or c, respectively. In a preferred embodiment, the affinity of the indicated variants for the fcrn (brambell) receptor is altered to fine tune its serum tolerance. In some embodiments, one or more histidine residues in positions H535, H510, H464 of these domains are deleted or substituted with another basic or non-basic amino acid. In some embodiments, the domain III sequence has or has only one or more substitutions of H535A or G, H510A or G, H464A or G. In some further embodiments, the substitution is 1, 2, or 3 of H535A, H510A, H464A. In other embodiments, the substitution is H535V, I, or L; H510V, L or I; or one or more of H464V, L, or I. In further embodiments, there are other conservative substitutions (1, 2, 3,4 or more) at other positions of the domain III, IIIa or IIIb scaffold. GenBank: AAA98797.1 also gives the sequence for HSA.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. Methods for obtaining (e.g., producing, isolating, purifying, synthesizing, and recombinantly producing) a polypeptide are well known to those of ordinary skill in the art.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Preferred amino acids are naturally occurring amino acids as found in humans. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids, such as hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. "amino acid analog" refers to a compound having the same basic chemical structure as a naturally occurring amino acid, i.e., an alpha carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but remain the same as the basic chemical structure of the naturally occurring amino acids. Amino acid mimetics refers to compounds that differ in structure from the ordinary chemical structure of an amino acid, but function in a manner similar to a naturally occurring amino acid.

Amino acids herein may be referred to by the universal three letter code or one letter code as recommended by the IUPAC-IUB Biochemical nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

With respect to "conservatively modified variants" of an amino acid sequence, one skilled in the art will recognize that a single substitution, deletion or addition of a nucleic acid, peptide, polypeptide or protein sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" wherein the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

The following eight groups each contain amino acids that may be conservatively substituted for each other: 1) alanine (a), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C) methionine (M) (see, for example, Creighton, Proteins (1984)).

From Hollinger et al, PNAS (USA)90 (14): 6444-6448(1993) the first diabody described can be constructed using the heavy and light chains described herein, and the individual CDR regions described herein can also be used. Diabody fragments typically comprise a light chain variable domain (V) joined by a linker_L) Linked heavy chain variable domains (V)_H) The linker is too short to pair between two domains on the same strand. Thus, V of a segment is forced_HAnd V_LV of Domain to Another fragment_HAnd V_LThe domains are complementary, thereby forming two antigen binding sites. Tri-antibodies with 3 antigen binding sites can be constructed similarly. The Fv fragment contains the entire antigen binding site, includingV held together by covalent interactions_LDomains and V_HA domain. The Fv fragments encompassed by the present invention also include cross-linking of V by glutaraldehyde, intramolecular disulfide bonds, or other linkers_HAnd V_LA domain construct. The variable regions of the heavy and light chains are fused together to form a single chain variable fragment (scFv) that retains the original specificity of the parent immunoglobulin. Single chain fv (scFv) dimers consisting of Gruber et al, J.Immunol.152 (12): 5368-74(1994), for the first time, can be constructed using the heavy and light chains described herein, as can the individual CDR regions described herein. Many techniques known in the art can be used to prepare the specific binding constructs of the present invention (see U.S. patent application publication No. 20070196274 and U.S. patent application publication No. 20050163782, each of which is incorporated by reference herein in its entirety for all purposes, particularly with respect to miniantibody and diabody designs).

Bispecific antibodies can be produced using hybridoma technology for chemical cross-linking or hybridization. Alternatively, bispecific antibody molecules can be produced using recombinant techniques (see: bispecific antibodies). Will connect to V_HAnd V_LDimerization may be promoted by shortening the linker length of the domains from about 15 amino acids, which are commonly used to generate scFv fragments, to about 5 amino acids. These linkers facilitate inter-chain assembly of the VH and VL domains. SGGGS is a suitable short linker, but other linkers may be used. The two fragments thereby assemble into a dimeric molecule. Further reduction of linker length to 0-2 amino acids can result in trimeric (triabody) or tetrameric (tetrabody) molecules.

Antibodies such as recombinant, Monoclonal or polyclonal Antibodies can be prepared using a number of techniques known in the art (see, e.g., Kohler and Milstein, Nature 256: 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, Monoclonal Antibodies and cancer therapy (Monoclonal Antibodies and cancer therapy), ARL Co., Ltd. (Alan R.Liss, Inc.), pp.77-96 (1985); Coligan, Current Protocols in Immunology (1991); Harlow and Lane, Antibodies, A Laboratory Manual (Antibodies: Laboratory Manual) (1988); and Goding, Monoclonal Antibodies: Principies and practice (1986); Monoclonal Antibodies and practice) principle). Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells, for example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Libraries of genes encoding heavy and light chains of monoclonal antibodies can also be made from hybridomas or plasma cells. Random combinations of heavy and light chain gene products produce large numbers of antibodies with different antigen specificities (see, e.g., Kuby, Immunology (3 rd edition, 1997)). Techniques for the production of single chain antibodies or recombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No. 4,816,567) can be adapted for the production of antibodies to the polypeptides of the invention. Transgenic mice or other organisms such as other mammals can also be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, Marks et al, Bio/Technology 10: 779-783 (1992); Lonberg et al, Nature 368: 856-859 (1994); Morrison, Nature 368: 812-13 (1994); Fishwild et al, Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern.Rev. Immunol.13: 65-93 (1995)). Alternatively, phage display techniques can be used to identify antibodies and heteromeric Fab fragments that specifically bind to the selected antigen (see, e.g., McCafferty et al, Nature 348: 552-78554 (1990); Marks et al, Biotechnology 10: 779-783 (1992)). Antibodies can also be made bispecific, i.e., capable of recognizing two different antigens (see, e.g., WO 93/08829, Traunecker et al, EMBO J.10: 3655-3659(1991) and Suresh et al, Methods in Enzymology 121: 210 (1986)). The antibody can also be a heterologous conjugate, e.g., two covalently bound antibodies or immunotoxins (see, e.g., U.S. Pat. Nos. 4,676,980, WO 91/00360, WO 92/200373; and EP 03089).

Methods for humanizing or primatizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as import residues, and are typically taken from an import variable region. Humanization can be carried out essentially as described by Winter and co-workers (see, e.g., Jones et al, Nature 321: 522-525 (1986); Riechmann et al, Nature 332: 323-327 (1988); Verhoeyen et al, Science 239: 1534-1536(1988) and Presta, curr. Op. struct. biol. 2: 593-596(1992)), by substituting rodent CDRs or CDR sequences for the corresponding human antibody sequences. Thus, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) in which significantly less than the entire human variable region is replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from similar sites in rodent antibodies.

A "chimeric antibody" is an antibody molecule in which (a) the constant regions or portions thereof are altered, replaced, or exchanged so that their antigen binding sites (variable regions) bind to constant regions that differ in type, effector function, and/or species or have been altered, or to completely different molecules that confer novel properties to the chimeric antibody, e.g., enzymes, toxins, hormones, growth factors, drugs, etc.; or (b) altering, replacing or exchanging the variable region or a portion thereof with a variable region having a different or altered antigenic specificity.

The term "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with … … in reference to a protein or peptide refers to the ability to determine the presence or absence of a binding reaction for the protein in a heterogeneous population of proteins and other biological substances. Thus, under the specified immunoassay conditions, a particular antibody binds to a particular protein at least twice, more typically 10-100 times or more, that of the background. Specific binding to antibodies under these conditions requires the selection of antibodies specific for the particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection can be achieved by subtracting out antibodies that cross-react with other molecules. Antibodies specifically immunoreactive with a particular protein may be selected using a variety of immunoassay formats. For example, solid phase ELISA immunoassays are routinely used to select Antibodies specifically immunoreactive with a protein (see, e.g., Harlow and Lane, Using Antibodies, A laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer that affects the transcription of a coding sequence is operably linked to the coding sequence; or a ribosome binding site is operably linked to the coding sequence at a position that facilitates translation of the coding sequence. Generally, "operably linked" refers to DNA sequences that are linked adjacent to each other and, in the case of secretory leader sequences, adjacent to each other and in reading phase. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. Without such sites, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

"conservatively modified variants" applies to both amino acid and nucleic acid sequences. For a particular nucleic acid sequence, conservatively modified variants refers to nucleic acids that encode identical or substantially identical amino acid sequences, or, when the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at each position where an alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variations. Each nucleic acid sequence herein encoding a polypeptide also describes each possible silent variation of that nucleic acid. One skilled in the art will recognize that individual codons in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce functionally identical molecules. Thus, each silent variation of a nucleic acid encoding a polypeptide is implicit in each sequence described for the expression product, but not to the actual probe sequence.

The term "identical" or percent "identity" with respect to two or more nucleic acid or polypeptide molecule sequences refers to two or more sequences or subsequences that are the same over a specified region or have a specified percentage of amino acid residues or nucleotides therein that are the same, as determined using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters described below, or by manual alignment or visual inspection (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity when compared and aligned for maximum correspondence over a comparison window or designated region) (see, e.g., NCBI website http:// www.ncbi.nlm.nih.gov/BLAST/etc.). These sequences may then be said to be "substantially identical". This definition also refers to, or is applicable to, the complement of the test sequence. The definition also includes sequences containing deletions and/or additions, as well as sequences containing substitutions. As described below, the preferred algorithm may take into account gaps and the like. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50-100 amino acids or nucleotides in length.

For sequence comparison, one sequence is typically used as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, both test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, as preferred, or additional parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

As used herein, a "comparison window" includes reference to a segment selected from 20 to the full length of a reference sequence, typically from about 25 to 100, or from 50 to about 150, more typically from about 100 to about 150, contiguous positions, and, when optimally aligned, a sequence can be compared to a reference sequence having the same number of contiguous positions. Methods of comparing aligned sequences are well known in the art. Can be determined, for example, by the local homology algorithm of Smith and Waterman, adv. 482(1981), homology alignment by Needleman and Wunsch, j.mol.biol.48: 443(1970) by the similarity search method of Pearson and Lipman, proc.nat' l.acad.sci.usa 85: 2444(1988), by Computer execution of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package of the Genetics Computer Group (Wisconsin Genetics Software Package), Madison, Wis.), or by manual alignment with visual inspection (see, for example, Current Protocols in molecular biology laboratory Manual (eds. Ausubel et al, 1995 supplement)) for comparison.

Preferred examples of algorithms suitable for determining sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively, consisting of Altschul et al, Nuc. acids Res.25: 3389-: 403-. Using BLAST and BLAST 2.0, the parameters described herein are set to determine the percent sequence identity of the nucleic acids and proteins of the invention. Software for performing BLAST analysis is publicly available from the national center for Biotechnology Information (http:// ncbi. nlm. nih. gov. /). The algorithm comprises the following steps: high scoring sequence pairs (HSPs) are first identified by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbor score threshold (Altschul et al, supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits extend in both directions along each sequence as long as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores were calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hits in all directions is aborted: the cumulative comparison score decreases by X from the maximum obtained value; the cumulative score becomes zero or below zero due to the accumulation of one or more negative-scoring residue alignments; or to the end of each sequence. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The default values employed by the BLASTN program (for nucleotide sequences) are as follows: word length (W)11, expected value (E)10, M-5, N-4, and compare both chains. For amino acid sequences, the BLASTP program uses default values of: word length 3, expectation (E)10, BLOSUM62 scoring matrix (see Henikoff and Henikoff, proc. natl. acad. sci. usa 89: 10915(1989)), alignment (B)50, expectation (E)10, M-5, N-4, and both strands.

"nucleic acid" refers to deoxyribonucleic or ribonucleic acids, as well as polymers thereof in single-or double-stranded form, and the complementary strands thereof. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring, and non-naturally occurring, that bind similarly to a reference nucleic acid, and that metabolize in a manner similar to that of the reference nucleotide. Examples of such analogs include, but are not limited to: phosphorothioate, phosphoramidate, methylphosphonate, chiral-methylphosphonate, 2-O-methyl ribonucleotide, peptide-nucleic acid (PNA).

Unless otherwise indicated, a particular nucleic acid sequence also includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19: 5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-2608 (1985); Rossolini et al, mol.cell.Probes 8: 91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The specific nucleic acid sequence also implies "splice variants". Similarly, a particular protein encoded by a nucleic acid also implies a protein encoded by a cleaved variant of the nucleic acid. As the name suggests, a "splice variant" is the product of alternative splicing of a gene. Following transcription, the initial nucleic acid transcript may be spliced such that different (alternative) nucleic acid splice products encode different polypeptides. The mechanism by which splice variants are produced varies, but includes alternative splicing of exons (alternative splicing). The definition also includes variant polypeptides (alternate polypeptides) produced by read-out transcription of the same nucleic acid. Included in this definition are any products of a splicing reaction, including recombinant forms of the spliced products. Leicher et al, j.biol.chem.273 (52): 35095-35101(1998) discusses examples of splice variants of the potassium channel.

The term "heterologous" when used in reference to a nucleic acid moiety means that the nucleic acid comprises two or more subsequences that are not in the same relationship as they are naturally found. For example, the nucleic acids are typically recombinantly produced, arranging two or more sequences from unrelated genes into a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein means that the protein comprises two or more subsequences that are not in the same relationship in nature (e.g., a fusion protein).

The term "stringent hybridization conditions" refers to conditions under which a probe hybridizes to its target subsequence, but not to other sequences, typically in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. For extensive guidance on Nucleic acid Hybridization, see Tijssen, Techniques in Biochemistry and molecular biology- -Hybridization with Nucleic acid Probes, Overview of Biochemistry of Hybridization and the strategy of Nucleic acid assays ("Hybridization principles and protocols for Nucleic acid assays") (1993). Generally, stringent conditions are selected to be about 5-10 ℃ lower than the thermal melting point (Tm) for the particular sequence at a defined ionic strength, pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the target complementary probe hybridizes at equilibrium with the target sequence (due to the excess target sequence, 50% of the probe is occupied at Tm at equilibrium). Destabilizing agents such as formamide may also be added to achieve stringent conditions. In selective or specific hybridization, the positive signal is at least twice, preferably 10 times, that of the background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃ or 5 XSSC, 1% SDS, incubated at 65 ℃ and washed with 0.2 XSSC and 0.1% SDS at 65 ℃.

If the nucleic acids encode substantially the same polypeptide, nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical. This may occur, for example, when a copy of a nucleic acid is produced using the maximum codon degeneracy permitted by the genetic code. In this case, the nucleic acid generally hybridizes under moderately stringent hybridization conditions. Exemplary "moderately stringent hybridization conditions" include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ℃ and washing in 1 XSSC at 45 ℃. Positive hybridization was at least twice background. One of ordinary skill in the art will readily recognize that other hybridization and wash conditions can be used to provide conditions of similar stringency. Many documents provide additional principles for determining hybridization parameters, such as Current protocols in Molecular Biology (New Molecular Biology laboratory Manual), eds Ausubel et al, John Wiley & Sons.

For PCR, the temperature for low stringency amplification is typically about 36 ℃ but the annealing temperature may vary from 32 ℃ to 48 ℃ depending on the primer length. For highly stringent PCR amplification, a typical temperature is about 62 ℃, but the annealing temperature may range from about 50 ℃ to about 65 ℃ depending on primer length and specificity. Typical cycling conditions for high and low stringency amplification include a denaturation phase of 30 seconds to 2 minutes at 90 ℃ to 95 ℃, an annealing phase lasting 30 seconds to 2 minutes, and an extension phase of 1 to 2 minutes at 72 ℃. For example, in Innis et al (1990) PCR Protocols, A Guide to Methods and Applications (PCR Protocols: guidelines for Methods and Applications, academic Press GmbH, N.Y.), Methods and principles for low and high stringency amplification reactions are provided.

A "label" or "detectable moiety" or "imaging agent or moiety" is a compound that is detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, radiological or other physical means. For example, availableIs marked by³²P, a fluorescent dye, an electron-dense reagent, an enzyme (e.g., an enzyme commonly used in ELISA), biotin, digoxigenin, or a hapten and a protein, which hapten or protein can be detected or used to detect an antibody specifically reactive with the peptide, for example, by incorporating a radioactive label into the peptide. Preferred imaging agents or moieties are magnetic, fluorescent or radioactive. Methods for detecting the signal generated by the label in vitro and in vivo are well known in the art.

The term "recombinant" when used with respect to, for example, a cell or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells thus express genes that are not present within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

A composition is provided.

For pharmaceutical purposes of the present invention, the constructs of the present invention are typically formulated in a suitable buffer, which may be any pharmaceutically acceptable buffer, such as phosphate buffered saline or sodium phosphate/sulfate, Tris buffer, glycine buffer, sterile water and other buffers known to those of ordinary skill in the art, such as Good et al, Biochemistry 5: 467 (1966). The compositions may also include stabilizers, enhancers or other pharmaceutically acceptable carriers or vehicles. A pharmaceutically acceptable carrier may comprise a physiologically acceptable compound, for example, for stabilizing a nucleic acid or polypeptide of the invention and any associated vector. For example, physiologically acceptable compounds may include carbohydrates such as glucose, sucrose or dextran; antioxidants such as ascorbic acid or glutathione; a chelating agent; low molecular weight proteins or other stabilizers or excipients. Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents, or preservatives, preservatives being particularly useful for preventing microbial growth or action. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of such carriers, stabilizers or adjuvants are described in Remington's Pharmaceutical Sciences (Remington Pharmaceutical Sciences), Mack Publishing Company (Mack Publishing Company), Philadelphia, Pa., 17 th edition (1985).

The pharmaceutical compositions of the invention comprise a therapeutically effective amount of the construct of the invention and a pharmaceutically acceptable carrier. As used herein, a "therapeutically effective dose or amount" refers to an amount that produces the desired effect of administering the composition (e.g., treating or preventing retinal detachment). The exact Dosage and formulation will depend on The purpose of The treatment, and can be determined by one skilled in The art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Pharmaceutical Dosage Forms) (Vol.1-3, 1992); Lloyd, TheArt, Science and Technology of Pharmaceutical Compounding (Art, Science and Technology of Pharmaceutical Compounding) (1999); Remington: The Science and Practice of Pharmacy (Remington: Pharmaceutical Science and Practice), 20 th edition, Gennaro eds. (2003), and Pickarr, Dosage Calculations (1999)). If the construct is a salt, the construct is formulated as a "pharmaceutically acceptable salt".

"pharmaceutically acceptable salts" include salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the route of administration. When the inhibitors of the invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of the compound with a sufficient amount of the desired acid, either in the pure acid form or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include: salts derived from inorganic acids such as hydrochlorides, hydrobromides, nitrates, carbonates, bicarbonates, phosphates, hydrogenphosphates, dihydrogenphosphates, sulfates, hydrogensulfates, hydroiodides or phosphites and the like, as well as salts derived from relatively nontoxic organic acids such as acetates, propionates, isobutyrates, maleates, malonates, benzoates, succinates, suberates, fumarates, lactates, mandelates, phthalates, benzenesulfonates, p-toluenesulfonates, citrates, tartrates, methanesulfonates and the like. Also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the invention contain both basic and acidic functional groups that are capable of converting the compound to a base or acid addition salt.

The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. Certain physical characteristics of the parent form of the compound, such as solubility in polar solvents, are different from the various salt forms, but in other aspects for purposes of the present invention the salts are equivalent to the parent form of the compound.

In addition to salt forms, the present invention also provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir containing a suitable enzyme or chemical agent.

Certain compounds of the present invention may exist in non-solvated as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are intended to fall within the scope of the present invention. Certain compounds of the present invention may exist in polycrystalline or amorphous form. In general, all physical forms are equivalent in the applications contemplated by the present invention and are intended to be within the scope of the present invention.

In addition to biopolymers such as nucleic acids and polypeptides, certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers and individual isomers are all intended to fall within the scope of the present invention. In preferred embodiments, the compound comprises amino acids or nucleic acids that are each their naturally occurring major biological enantiomer

The compositions administered will generally comprise the agent dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier, as described herein. A variety of aqueous carriers can be used, such as buffered saline and the like. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to simulate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on factors such as fluid volume, viscosity, body weight, etc., according to the particular mode of administration selected and the needs of the patient.

Suitable formulations for use in the present invention are described in Remington, incorporated herein by reference: the science and Practice of Pharmacy (Remington: science and Practice of medicine), 20 th edition, eds (2003). In addition, a brief review of drug delivery methods is found in Langer, Science 249: 1527 and 1533(1990), which are incorporated herein by reference. The pharmaceutical compositions described herein may be manufactured in a manner known to those skilled in the art, i.e., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. The following methods and excipients are exemplary only and are not intended to be limiting in any way.

For injection, the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as hanks (Hank's) solution, Ringer's solution or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the inhibitors for use in the present invention may be conveniently formulated by combination with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by: the compounds are mixed with solid excipients, optionally grinding the resulting mixture, and processing the mixture, if desired after addition of suitable auxiliaries, into granules to obtain tablets or dragee cores. In particular, suitable excipients are fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations are, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, for example cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

The pharmaceutical compositions can be administered in a variety of dosage forms and amounts depending on the method of administration. For example, unit dosage forms suitable for oral administration include, but are not limited to, powders, tablets, pills, capsules, lozenges. It is recognized that when the antibody is administered orally, it should be protected from digestion. This is usually done by complexing the molecule with a composition that makes it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule within a suitable resistant carrier such as a liposome or protective barrier. Methods of protecting agents from digestion are well known in the art.

In particular, pharmaceutical formulations of the constructs of the invention may be prepared by mixing the construct of the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer. These formulations may be lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. The acceptable carrier, excipient or stabilizer may be acetic acid, phosphoric acid, citric acid or other organic acid; antioxidants (e.g., ascorbic acid); a preservative; a low molecular weight polypeptide; proteins, such as serum albumin or gelatin, or hydrophilic polymers such as polyvinylpyrrolidone; and amino acids, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; a chelating agent; and ionic and nonionic surfactants (e.g., polysorbates); salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or a nonionic surfactant. In some embodiments, the construct may be formulated at a concentration of between 0.5-200mg/ml or 10-50 mg/ml.

Compositions comprising the constructs of the invention may be administered for diagnostic, therapeutic or prophylactic treatment. In therapeutic applications, the compositions are administered to a patient in a "therapeutically effective dose". The compositions may be administered in single or multiple administrations depending on the dosage and frequency required and tolerated by the patient. "patients" or "subjects" for the purposes of the present invention include humans and other animals, particularly mammals. Thus, the methods are suitable for human therapy and veterinary applications. In a preferred embodiment, the patient is a mammal, preferably a primate, and in a most preferred embodiment, the patient is a human.

The term "carrier" as used herein refers to a typically inert material used as a diluent or carrier for an active agent (e.g., a drug such as a therapeutic agent) to be used in a biological system, either in vivo or in vitro. The term also covers typical inert substances that impart adhesive properties to the composition.

The compositions of the present invention may be sterilized by conventional, well known sterilization techniques or may be manufactured under sterile conditions. The aqueous solution is packaged for use as is, or filtered under sterile conditions and lyophilized, the lyophilized formulation being mixed with a sterile aqueous solution immediately prior to administration. The compositions may contain pharmaceutically or physiologically acceptable auxiliary substances as required to simulate physiological conditions such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate.

Method of treatment

The term "treating" or "treatment" includes:

(1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to the organism but has not yet developed or displayed symptoms of the disease,

(2) inhibiting the disease, i.e., arresting or slowing the progression of the disease or its clinical symptoms. This includes reducing the degree of observed shedding or the number of objects or the risk of shedding of objects.

(3) Alleviating the disease, i.e., causing a decline in the disease, or its clinical symptoms.

The constructs for use according to the invention may be administered by any route of administration (e.g., intravenous, topical, intraperitoneal, parenteral, oral, intravaginal, rectal, ocular, intravitreal, and intraocular). They may be administered as a bolus, or by continuous infusion over a period of time, by intramuscular, intraperitoneal, subcutaneous, oral, topical or inhalation routes. Intravenous or subcutaneous administration of the antibody is preferred. The administration may be local or systemic. They can be administered to a subject diagnosed with, having a history of, or at risk of developing a disease.

Examples

The following examples are provided to illustrate, but not to limit, the claimed invention. The present invention is exemplified by a fusion protein of CEA and a domain III scaffold (see FIG. 8), while other methods of coupling the scaffold to a targeting agent and or imaging to a therapeutic agent are also contemplated.

Example 1: we tested fusion proteins consisting of a well-studied antibody fragment targeting carcinoembryonic antigen (CEA) and hsadii wild type (WT, no mutation) or one of three HSA DIII variants, each having a mutation of H535, H510, or H464 to an alanine residue. By using¹²⁴I-labeled Db-DIII or Db protein injection xenograft athymic nude miceMouse, a series of small animal PET/CT imaging studies were performed to assess the ability of HSA DIII to modulate Db persistence in vivo. Furthermore, we can conclude on the relative importance of H535, H510 and H464 residues for FcRn binding and circulation persistence of albumin.

Materials and methods

Generation of Db-DIII constructs

The HSA DIII gene was amplified by Polymerase Chain Reaction (PCR) using commercially available HSA cDNA (OG technologies, Rockwell, Md.) as template and primers to introduce a 5 'SpeI and 3' EcoRI restriction site. The primer sequences are as follows:

forward direction: SpeI-DIII: 5'-CCACTAGTGGCGAAGAGCCTCAGAATTTAATC-3'

And (3) reversing: DIII-EcoRI: 5'-GAGAATTCTATTATAAGCCTAAGGCAGCTTGAC-3'

Mutations to alanine at histidine residues H535, H510 or H464 were introduced in DIII by site-directed mutagenesis using the Quick-Change mutagenesis kit (statsuba corporation (Stratagene), lahoa, california), using appropriate mutagenesis primers (only the forward primers listed):

H464A (histidine residue at position 464 was exchanged with alanine residue)

5’-CTGAACCAGTTATGTGTGTTGGCTGAGAAAACGCCAGTAAGTGAC-3’

H510A

5’-GTTTAATGCTGAAACATTCACCTTCGCTGCAGATATATGCACAC-3’

H535A

5’-CTGCACTTGTTGAGCTCGTGAAAGCCAAGCCCAAGGCAAC-3’

The complete DIII (WT, H535A, H510A and H464A) genes were cloned into pCR2.1-Topo vector (Invitrogen, Calif.) and then transferred into pUC18 vector (NEB, New England Biolabs, Bevolley, Mass.) which already contains anti-CEA Db (Wu et al, 1999). The entire Db-DIII gene was excised from the pUC18 vector using XbaI and EcoRI sites and ligated into the pEE12 mammalian expression vector (Bebbington et al, 1992).

Expression, screening and purification

NS0 mouse myeloma cells (Sigma-Aldrich, St. Louis, Mo.) were maintained in nonselective glutamine-free Dulbecco's modified Eagle medium (DME/highly modified, SAFC Biosciences (SAFC Biosciences, Kansas, Lei.) supplemented with 5% heat-inactivated, dialyzed fetal bovine serum (FBS; Omega Scientific Inc.), Taza, Calif.), 1% v/v of 200mM L-glutamine (Meditech, Inc.), Manassas, Va.) and 1% v/v of penicillin-streptomycin (10,000IU/ml penicillin, 10,000 μ g/ml streptomycin, Miditatco, Inc.). Transfection of 1X10 in log phase growth with 10. mu.g of pEE12-Db-DIII DNA by electroporation⁷NS0 cells, the DNA was linearized by SalI (NEB, Ipsweiqi, Mass.) digestion as described previously (Kenanova et al, 2005).

Db-DIII production was determined by ELISA and confirmed by Western blotting. Protein A (Thermo Fisher Scientific, Rockford, Ill.) was used to capture Db-DIII protein in an ELISA. Alkaline Phosphatase (AP) -conjugated anti-mouse Fab-specific antibodies (sigma-aldrich) were used for detection in both ELISA and Western blots. Transfected NS0 cells were maintained in selective glutamine-free DME/highly modified medium (SAFC biosciences) supplemented with 5% heat-inactivated dialyzed FBS (omega science, Inc.), 2% v/v 50 XGS supplement (SAFC biosciences), and 1% v/v penicillin-streptomycin (Midiyazaitake, Inc.). Selected clones expressing the Db-DIII protein in large quantities were expanded stepwise to 3 flasks (Nunclon, Rockchester, N.Y.), each containing 300ml of selective medium supplemented with 2% heat-inactivated dialyzed FBS (omega science, Inc.) and 1% v/v penicillin-streptomycin (Midiitake, Inc.).

When the culture reached the end stage (. about.3 weeks), the collected supernatant was centrifuged through a molecular weight cut-off (mwco)30,000Da filter, sterile filtered and concentrated using a laboratory grade Tangential Flow Filtration (TFF) system (Millipore, Billerica, Mass.). Db-DIII protein was purified on a protein A column (Selmo Fisher science, Inc.) using an AKTA purifier (GE Healthcare, Pescat Vicat, N.J.). Bound protein was eluted with 15% 0.2M citrate buffer (pH 2.1) in 1 XPBS and 80% v/v 1M Tris base (pH 8.2) was added directly to the eluted protein to immediately neutralize the pH. The fractions containing the Db-DIII protein were combined, dialyzed against 1 XPBS, and concentrated with Vivaspin 20 (mwco: 30,000; S-S Biotech GmbH (Sartorius StedimBiotech Gmbh), Geltin, Germany). With A₂₈₀The final concentration of purified Db-DIII protein was measured, and the extinction coefficient ∈ was 1.5.

Identification of Db-DIII proteins

Purified Db-DIII protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, mass spectrometry, and size exclusion chromatography under both non-reducing (NR) and reducing (R) conditions. To reduce the protein, 1M Dithiothreitol (DTT) was added to a final concentration of 0.2M. For SDS-PAGE, 4-20% gradient Tris-HCl ready-to-use gels (Bio-Rad Laboratories, Inc.), Helercus, Calif.) were run and developed with transient Coomassie Blue based Solutions (EPS corporation, expedienon proteins Solutions, Cambridge, UK). Detection of Db-DIII protein in Western blots was achieved by AP-conjugated goat anti-mouse Fab-specific mabs (sigma-aldrich) using Nitrocyanobenzo (NBT) and 5-bromo-4-chloro-3-indole-phosphate (BCIP) (Promega, madison, wisconsin) AP substrate or with horseradish peroxidase (HRP) -conjugated protein L (sigma-aldrich) and developed with 4-chloro-1-naphthol/3, 3' -diaminobenzidine (CN/DAB) substrate kit (Thermo Scientific, rockford, illinois).

Mass spectrometry was performed using a LTQ-FT ultra-linear ion trap Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Selmore Fisher Co.) to confirm the identity of the purified protein. Briefly, Db-DIII protein was isolated and then subjected to an In-Gel tryptic digestion process, which is detailed In http:// massspec.wiki.zo.com/In-Gel-Trypsin-digest.html. Nano liquid chromatography tandem mass spectrometry (nLC-MSMS) and Collision Activated Dissociation (CAD) segmentation was performed on LTQ-FT (sefmorel) integrated with ekstrigennano-LC. Maps were searched against the latest international protein index database (version 3.54, with 39,925 entries) using Mascot (Matrix Science, UK) and sequence (semarfischer) programs. The results were filtered using a strict scoring filter criteria and a 10ppm mass resolution filter. The identified peptides also matched the Db-DIII sequence.

Protein purity of the purified Db-DIII, protein conformation of the Db-DIII in native, non-denaturing conditions (1 × PBS, pH 7.4), and molecular size assessment were determined by size exclusion chromatography using a Superdex 200 HR 10/30 column (GE healthcare).

Computer models of DIII and Db-DIII molecules were generated using PyMOL software (DeLano Scientific). Furthermore, modeling of the protein interface between HSA and DIII was achieved with the ZDCK-FFT algorithm (Chen et al, 2003), which is available on a common server (http:// zlab. bu. edu/rong/dock).

Radioiodination, xenograft imaging, and biodistribution of Db-DIII fusion proteins

Purified Db-DIII WT, H535A, H510A and H464A were obtained by positron emitters¹²⁴I (sodium iodide in 0.02M NaOH; IBA molecular Co., Stirling, Va.) was radioiodinated using the Iodogen method described previously (Olafsen et al, 2006). The labeling reaction (0.114-0.130ml) contained 0.1mg of purified protein and 12.9-18.0MBq Na¹²⁴I. Monoclonal antibody ITLC strip kit (BMS, Biodex Medical Systems, Xueli, N.Y.) was used as described previously (Olafsen et al, 2006)The labeling efficiency was determined by Instant Thin Layer Chromatography (ITLC).

For in vivo studies, 7-8 week old athymic nude mice (Charles river Laboratories, Wimington, Mass.) were injected subcutaneously with 1-5x10 in the left shoulder region⁶CEA-positive LS174T human colon cancer cells (American type culture Collection, Mass.) were injected in the right shoulder region with approximately the same number of CEA-negative C6 rat glioma cells (ATCC). The tumor mass was allowed to develop for an average of 10 days to a maximum of 200mg by weight. 3.9-5.4MBq for each construct¹²⁴4 tumor-bearing mice were injected intravenously with Db-DIII labeled I or Db tail in saline/1% HSA. At 5 different time points (4 hours, 20 hours, 28 hours, 44 hours, and 51 hours), the injected mice were anesthetized with 2% isoflurane, placed on the experimental bed, and imaged for 10 minutes. A 10 minute CT scan was completed after the 51 st hour final PET scan. All imaging experiments utilized Focus 220 small animal PET (Siemens preclinical Solutions, Nursery, Tenn.) and small animal CAT II (Concord Microsystems, Nursery, Tenn.) scanners. After the last scan (51 hours), mice were sacrificed. Blood, tumors (LS174T and C6), liver, spleen, kidney, lung and cadavers were collected, weighed and counted in a Wallac WIZARD automatic gamma counter (PerkinElmer Life and Analytical Sciences Inc., welssle, ma). After decay correction, the percent dose injected per gram (% ID/g) of each tissue/organ was calculated, including the correction for each protein labeling efficiency and standard deviation (SE).

Imaging analysis and statistics

All images were reconstructed with Filtered Backprojection (FBP) algorithm (Defreise et al, 1997) and rendered with AMID software (Loening and ambhir, 2003). The same color threshold is used for all images. A region of interest (ROI; oval, 0.4mm deep, n-4) was drawn within the CEA-positive tumor region and low activity, soft tissue regions (muscles) of the lower part of the body. Tumor to soft tissue (T: ST) ratios were determined for each mouse and averaged for each time point and construct. An ROI (n-4) was drawn on the heart of each image and the% ID/g of blood was calculated by the AMIDE software after the injection dose in MBq and cylinder factor in MBq/cc/image as input functions. The Mean Residence Time (MRT) of each protein was calculated from the blood activity curves using the ADAPTII software package (D' argenio and Schumitzky, 1979). SE was calculated for all ratios and% ID/g values and plotted (error bars). The unpaired student's T test was used to compare all T: ST ROI ratios and blood activity curves separately to determine significant differences. Two-sided P values below or equal to 0.05 were considered statistically significant.

Results

Production and biochemical identification of Db-DIII proteins

a. Production, expression and purification

The Db-DIII construct was approximately 1.4 kilobase pairs long, flanked by XbaI and EcoRI restriction sites (FIG. 1A). In the final culture of transfected NS0 cells, the engineered Db-DIII molecules were expressed at 10-16. mu.g/ml as measured by ELISA. Although protein L binds to the Db-DIII protein, capture of protein A is more efficient. Thus, protein a affinity chromatography was selected for purification. Db is a non-covalent dimer of two scFv molecules, each Db molecule having two DIII proteins bound to its C-terminus, so the fusion protein has a calculated molecular weight of about 101kDa (fig. 1B).

SDS-PAGE and Western blotting

Db-DIII WT and variants were purified by SDS-PAGE analysis under NR and R conditions (FIG. 2A). Under NR conditions, the Db-DIII protein produced a major band corresponding to its predicted molecular weight of about 101kDa (FIG. 2A, lanes 1, 2 and 3). Two weak bands of lower molecular weight were noted in both the SDS-PAGE Coomassie stained gel (FIG. 2A, lanes 1, 2 and 3) and the Western blot probed with anti-mouse Fab specific antibody (FIG. 2B, lane 2). In reduction, the main band [ (scFv-DIII)₂；101kDa]Separation into 2 bands corresponding to scFv-DIII fragments (. about.48 kDa) and DIII molecules (. about.23 kDa) (FIG. 2A, lane 5). Detection of the DIII portion of the fusion protein Using polyclonal anti-HSA antibodyThe split attempt was unsuccessful, so the Western blot was modified with HRP-conjugated protein L that bound the Db-DIII protein Db fraction (FIG. 2B, lane 3). Only the upper portion [ (scFv-DIII) was determined₂；101kDa]And a middle (scFv-DIII; 48kDa) band. Thus, the smaller band of about 23kDa on the reduced protein SDS-PAGE gel (FIG. 2A, lane 5) should represent only the DIII domain. To confirm that the purified protein was indeed Db-DIII, mass spectrometry was performed. Multiple peptides matching the Db or DIII amino acid sequences were identified, confirming the identity of the protein (data not shown).

c. Size exclusion chromatography

Size exclusion chromatography showed that Db-DIII WT (101kDa) eluted as a single peak with an elution time of 28.17 minutes (FIG. 2C). Under the same conditions, the Db-DIII H535A, H510A and H464A proteins were characterized by a mean elution time of 28.2 minutes, and no aggregation or multimerization was detected. Integration of the size exclusion chromatography peaks showed protein purity of about 98% after one-step protein a affinity column purification.

d. Computer simulation of HSA DIII, DIII-FcRn interaction and Db-DIII

A structural model of HSA DIII was generated from the crystal structure of HSA (Sugio et al, 1999) (FIG. 3A). DIII contains 10 alpha-helices (6 in DIIIa, 4 in DIIIb) connected to each other by loops. Residues H464 (in DIIIa), H535 and H510A (all in DIIIb) are indicated. The crystal structure of DIII and FcRn was also used (Martin et al, 2001) to generate a docking model for DIII and FcRn (fig. 3B). The ZDCK algorithm favours the interaction involving FcRn residues H161 and H166(Andersen et al, 2006) and HSADIII H535, H510 and H464. 11 candidate structures are generated. These structures were sorted and analyzed for possible strong pH-dependent binding using PyMOL. The overall impression of these analyses is that the conserved aromatic residues around FcRn residues H166 and H161 occur in most predicted structures and thus they are likely to contact both DIII H510 and H535 residues. FcRn H166 and H161 appear to potentially interact with glutamate residues on DIII, increasing affinity when histidine is protonated in low pH environments. In addition, FcRn residues D102 and N101 interact in many proposed structures, possibly playing a role. It is believed that the 10 th resulting structure provided by ZDOCK most likely shows strong pH-dependent binding. The structure contains DIII H535 and FcRn F157; DIII H510 with FcRnW51 and Y60; DIII H464 with FcRn D101 and N102 or K123; FcRn H166 and DIIIE 505; and possible interactions between FcRn H161 and DIII E531. Finally, the crystal structure of the T84.66 diabody (Carmichael et al, 2003) was used to generate a model for the Db-DIII molecule (FIG. 3C). Each diabody binds two DIII molecules via an 18 amino acid linker that will result in a relatively flexible molecular structure.

Radiolabeling and mouse xenograft imaging studies

Of Db-DIII fusion proteins¹²⁴The labeling efficiency of I was 63.9-81.5%, while the injection-specific activity was 13.0-18.0 GBq/. mu.mol. Series of small animal PET imaging allowed in vivo comparison of Db-DIII fusion protein with Db alone for tumor targeting and persistence in circulation (figure 4). The images show that all 5 proteins target LS174T (CEA positive) tumors. The tumor anatomical location is clearly visible on the CT image. Targeting was noted as early as 4 hours for Db and Db-DIII H464A, whereas Db-DIII WT, H535A and H510A molecules found targeting at 20 hours. For all proteins, the signal in CEA-positive tumors persisted throughout the study (51 hours), while the background (circulating activity) varied. Statistical comparison of the T: ST ROI ratios at different time points for Db-DIII and Db proteins (FIG. 5A) showed that at 4 and 20 hours, all proteins showed no significant difference in T: ST ratios from each other (P > 0.05). From 28 hours on, to 44-51 hours, the Db T: ST ratio remained significantly greater than all Db-DIII proteins (P values in the range of 0.04-0.01). At 51H, the H535A (P0.03), H510A (P0.02) and H464A (P0.01) variants showed significantly higher T: ST ratios than WT due to the faster rate of soft tissue signal attenuation. However, the ratio of H510A T: ST was not significantly different from H535A (P ═ 0.1). The H464A T: ST ratio was not significantly different from the H510A T: ST ratio (P ═ 0.07). The ratio of H464A T: ST was significantly different from H535A (P ═ 0.02). Quantitative analysis of PET images of radioactivity (% ID/g) in blood at each time point resulted in a blood activity curve (fig. 5B) and the MRT of each protein in blood was calculated (table 1). And the placeWith histidine mutants, Db-DIII WT showed significantly slower kinetics of blood clearance (P < 0.05) compared to Db alone. There was no significant difference in the blood activity curves of Db-DIII H535A and H510A, and H510A and H464A from each other (P0.09 and P0.08, respectively), but H535A was characterized by significantly longer blood persistence (P0.01) compared to the H464A variant. Thus, the serum MRTs are, in order from longest to shortest: Db-DIII WT > H535A ≧ H510A ≧ H464A > Db, where the cyclic residence time of Db-DIIIH535A is significantly extended over H464A. The biodistribution at 51 hours confirmed the ranking of serum persistence (table 2). The activity of Db-DIII protein measured in blood ranged from 4.0-1.6% ID/g, whereas LS174T tumor uptake ranged from 2.5-1.3% ID/g, compared to 0.5% ID/g uptake for Db. Previous studies showed that radioiodination of T84.66Db reached maximum tumor uptake 2 hours after injection (13.68. + -. 1.49% ID/g), after which the activity within the tumor began to decrease (Wu et al, 1999). The mean tumor volumes for LS174T and C6 tumors were 161mg and 126mg, respectively. Note that longer serum residence times are generally associated with higher LS174T tumor uptake. The ratio of CEA positive to negative tumor uptake for the Db-DIII protein ranged from 1.5 to 2.2, compared to 13 for Db alone at 51 hours.

TABLE 1 biodistribution, shown in descending order of protein circulation persistence (% ID/g) from top to bottom¹²⁴I labeled Db-DIII protein.

Note: each protein was analyzed in groups of 4 mice. Organ uptake was expressed as% ID/g.

Table 2 estimation of blood half-life of Db-DIII and Db in tumor-bearing athymic nude mice.

¹AUC is the area under the curve ^ ju (t) dt from 0 to infinity.²The first Mo is the first time: integral ^ t u (t) dt/[ integral ] u (t) dt, u (t) is the measured (% ID/g) blood curve. 3 mean residence time is the same integral form but u (t) is replaced by du/dt.

Discussion of the related Art

The first step is proof of the principle that HSA DIII can act as a tunable PK protein scaffold, we involved a fusion protein consisting of two components. One is anti-CEA T84.66Db, a small bivalent antibody fragment that has been extensively studied in vivo. The anti-CEA Db showed a terminal β half-life in LS174T (CEA positive) tumor-bearing mice in the range of 2.89 hours (¹²³I) 3.04 hours (¹¹¹In) (Yazaki et al, 2001). This Db has also been successfully fused to other proteins (i.e., Renilla or Gaussian (Gaussia) luciferase) and retains its in vivo targeting ability after fusion (Venisnik et al, 2007; Venisnik et al, 2006). Thus, the Db is a good model for proof of concept studies of targeting molecules. The second component has an unknown characteristic, named HSA DIII WT or a variant thereof having a mutated H535, H510 or H464 residue. The Db-DIII fusion protein is expressed in mammalian cells to ensure proper folding. The expression level was reasonable and the molecular weight of the protein obtained by affinity purification was consistent with the calculated 101kDa (FIG. 2A). Db is a non-covalent dimer of two scFv molecules that separate from each other and migrate approximately 25kDa under SDS-PAGE conditions (Wu et al, 1999). We expect the Db-DIII molecule to migrate as the scFv-DIII (. about.48 kDa) species because DIII pairs with all cysteine residues in the scFv (Curry et al, 1998; Dugaiczyk et al, 1982; Wu, 1999). Interestingly, under non-reducing conditions, the protein body retains its dimeric form [ (scFv-DIII)₂；101kDa]The structural stability is shown to be improved under SDS conditions. After close examination of the Db-DIH computer model (fig. 3C), the linker length between Db and DIII was shortened from 18 amino acids to 5 (data not shown). This change did not affect the migration pattern of the protein [ (scFv-DIII)₂(ii) a FIG. 2A]. Due to this unexpected manifestation, the Db-DIII protein band of the SDS-PAGE gel was excised (FIG. 2A), and the extracted protein was analyzed by mass spectrometryProtein (data not shown). The results confirmed that the protein of about 101kDa was indeed Db-DIII. The increased stability of SDS and heat may be the result of polar, ionic interactions between the two scFv-DIII molecules, as is the case with β -glycosylase (Gentile et al, 2002). Size exclusion chromatography under physiological conditions confirmed the molecular size of the Db-DIII protein. The Db-DIII elutes at a time close to that of another protein of similar molecular weight (scFv-Fc, 105kDa), which elutes at about 27.3 minutes under the same conditions (Kenanova et al, 2005). Slightly smaller Db-DIII eluted at about 28.2 minutes, while Db alone eluted at 38.2 minutes (Kenanova et al, 2005). In addition to high purity, a single peak on the chromatogram (fig. 2C) shows the integrity of the Db-DIII protein and shows its presence as a single species. Analysis of the DIII-FcRn docking model (fig. 3B) helped to identify possible FcRn interaction partners in H535, H510 and H464 and were able to determine their importance in FcRn binding. The actual ordering of DIII histidine residues was determined by in vivo molecular imaging.

Molecular imaging, particularly PET, has the advantage of being able to topologically image the same individual multiple times after tracer injection to obtain quantitative PK, tumor targeting, cross-reactivity information. In this study, CEA-loaded positive and negative xenograft-bearing mice received¹²⁴Either Db-DIII or Db protein was labeled I and injected and imaged at 5 different time points. This allows a one-to-one comparison of the circulating persistence and tumor targeting of each Db-DIII protein, as well as Db alone. Since Db is a constant component, the PK differences between Db-DIII proteins are due to DIII function. Thus, PET imaging enabled us to conclude on the in vivo performance of DIII proteins, although this was indirect. Targeting agents that clear more quickly from serum achieve higher T: ST ratios at earlier time points. Therefore, based only on the T: ST ROI ratios at the time points (FIG. 5A), we can deduce that the order of fastest (highest T: ST ratio) to slowest (lowest T: ST ratio) blood clearance is: db > Db-DIII H464A > H510A > H535A > WT. Interestingly, statistical analysis showed that Db-DIII H510A cleared no significantly faster than H535A. Both H535 and H510 residues are located in the DIIIb subdomain. This finding may suggest that the H510 and H535 residues within albumin may be redundant, one of which is non-functional or non-functionalCan play a backup role in the condition of combining with FcRn. This hypothesis is yet to be elucidated. Simultaneous mutation of both residues provides more insight. The same ordering of circulating clearance was also confirmed by the blood activity curves obtained from quantitative analysis of radioactivity in mouse hearts for each protein at each time point (fig. 5B). Statistical comparison of blood activity curves led to the same conclusions as above-Db-DIII H535A cleared significantly more slowly than H464A, but with no significant difference compared to H510A. Due to the lack of more time points within the first 12 hours after tracer injection, it is more feasible to calculate MRT rather than α and β half-lives. MRT ranged from about 2.4 days for Db-DIII WT to 17 hours for Db-DIII H464A, compared to 2.9 hours for Db alone. The overall size of the Db-DIII fusion protein (101kDa) exceeded the threshold for renal clearance (. about.60 kDa). Thus, the Db-DIII protein is eliminated via the hepatobiliary pathway, while Db (55kDa) is cleared by the kidney. The difference in molecular weight between Db and Db-DIII proteins is therefore the main reason for the difference in MRT. However, the fact that Db-DIII PK in vivo can be modulated by single amino acid mutations (of the same molecular weight) suggests that there are other molecular mechanisms that control serum PK in vivo (e.g., FcRn interactions) in addition to an increase in molecular size. Furthermore, mutations (H535A, H510A, or H464A) allow fine tuning of the overall protein serum residence time. Various circulatory half-lives can be selected from the range of days to hours. This facilitates screening for diagnostic or therapeutic agents required for a particular serum PK.

Imaging studies clearly show that the HSA DIII domain can improve the circulating persistence of Db while maintaining tumor targeting. The retention time of all Db-DIII proteins in blood was significantly longer than Db alone (P < 0.05). Direct counts of residual radioactivity (% ID/g) in blood of mice injected with Db-DIII protein at 51 hours resulted in 50-fold (WT) to 20-fold higher levels than in mice injected with Db (H464A). In summary, our findings indicate that HSA DIII WT and the mutant itself should be able to modulate the serum residence time of its binding moiety. The DIII ordering of blood clearance is expected to remain the same as the experimentally determined Db-DIII order. DIII WT also extended serum persistence of Db slightly more than that of intact HSA molecule for tb 84.66scfv (Yazaki et al, 2008). LS174T xenograft withoutInjection for nude mice with thymus gland¹²⁵The remaining activity in blood 48 hours after labeling the anti-CEA scFv-HSA fusion protein (. about.90 kDa) was 2.79% ID/g, in contrast to injection¹²⁴The remaining activity 51 hours after labeling of Db-DIII WT with I was 4.00% ID/g (Table 2). This difference may be explained by the larger molecular weight of Db-DIII. Nevertheless, DIII was shown to be necessary and sufficient to maintain the serum half-life of the intact HSA molecule. H464 (located in DIIIa) appears to have the greatest impact on FcRn binding and circulatory persistence. Furthermore, since the H535A and H510A mutations produce significantly faster serum clearance than WT, we believe that both subdomains DIIIa and DIIIb are involved in maintaining serum persistence.

The purpose of the Db-DIII protein is to demonstrate the potential of HSA DIII to act as a single domain scaffold with controlled PK. The expression of DIII WT and variants without targeting moieties and the assessment of their in vivo PK is the next step in the screening of DIII scaffolds with optimal properties for imaging or therapeutic applications. The DIII scaffolds described in this study can be used to graft or chemically couple tumor targeting molecules (peptides, aptamers, small chemical molecules) or directly generate combinatorial libraries for display. Targeted scaffolds with appropriate PK for imaging may be used for diagnostic purposes. Alternatively, potential anti-tumor drugs can be conjugated to targeting scaffolds with optimal characteristics for cancer therapy.

Example 2.

Conjugation of DIII proteins with Alexa Fluor 647 for binding studies

The fluorophore Alexa Fluor 647(1.25kDa) was coupled to HSA, DIIIWT, H535A, H510A, and H464A proteins using an Alexa Fluor 647 protein labeling kit (Invitrogen, ewing, oregon) according to the manufacturer's instructions. Dilutions (in triplicate) of each fluorescent protein in the range of 0.316-3160nM were incubated with fusion 293 human embryonic kidney cells expressing human FcRn (Petkova et al, Int Immunol.2006; 18: 1759-1769) in round bottom 96-well plates at pH 6.5. Dilutions of HSA coupled with Alexa Fluor 647 were also incubated with 293 cells not expressing FcRn (control reaction). After washing step with 1 XPBS (pH 6.5), cells were washed by Maestro^TMIn vivo fluorescenceThe light imaging system (CRi Inc., Wobbe, Mass.) uses deep red (671-705nm) excitation and red (700nm long range) emission filters for imaging. The same size region of interest (ROI) was drawn in each well, the fluorescence signal was measured and averaged for each dilution. The binding curve was generated as a function of the concentration of the DIII protein coupled with Alexa-Fluor 647 using the mean fluorescence. The DIII concentration at which 50% fluorescence is measured represents the relative binding affinity of the DIII protein for FcRn (see figure 6).

DIII proteins bind to human FcRn

FIG. 7 shows that the more left-shifted binding curves for HSA and DIII protein coupled to the fluorophore (HSA and DIII WT) indicate stronger binding to FcRn-expressing 293 cells in the range of 100nM relative binding affinity, followed by DIII H535A and H510A (200 and 300nM, respectively), and the lowest relative binding affinity of DIII H464A, about 1. mu.M. HSA coupled with Alexa Fluor 647 did not bind 293 cells (FcRn-free) and thus demonstrated specific interaction with FcRn. Based on cell binding studies, the order of high to low binding affinity to human FcRn is as follows: HSA > DIII WT > DIII H535A > DIII H510A > DIII H464A.

Circulating half-life of DIII protein in mice

Of HSA and DIII proteins¹³¹The labeling efficiency of I is 39.6-93.6%, while the injection specific activity is 1.5-3.1. mu. Ci/. mu.g. The blood activity curves (FIG. 10) for intact HSA and all DIII proteins show the same elimination sequence as observed for the Db-DIII fusion protein, but with the addition of DIIIa and DIII. Furthermore, the decrease in relative binding affinity of HSA and DIII for FcRn (fig. 7) is proportional to the decrease in circulating persistence. Table 3 summarizes the estimates of blood half-life. The sequence of blood clearance from slow to fast is as follows: HSA > DIII WT > DIII H535A > DIII H510A > DIII H464A > DIIIa > DIIIb. The slow phase (β) half-life spans about 2-fold between proteins from the slowest (DIII WT) to the fastest clearance (DIIIb)_1/2Beta ranges from 15.3 to 6.9 hours. This range of cycle dwell times allows selection of the DIII platform that best suits the desired application (e.g., treatment, imaging).

TABLE 3 estimation of blood half-life of HSA and DIII proteins in Balb/c mice.

¹The amplitudes of the two components are given by A α and A β, where the sum of A α and A β is the total% ID/g.²t_1/2α＝ln2/k1，t_1/2β＝ln2/k2。³The area under the curve (AUC) is the time integral of blood uptake (% ID/g x h).

Example 3.

Aptamer molecules for screening DIII scaffolds were generated and coupled. Modified target-specific aptamers comprising nuclease-resistant pyrimidine 2 '-fluoro UTP and 2' FCTP can be produced by uncontrolled transcription of a double-stranded DNA template comprising the T7 RNA polymerase promoter. The transcription reaction can be performed using the Y639F mutant of T7 RNA polymerase. The nucleotides used in the reaction consisted of ATP, GTP, 2 'F dCTP and 2' F dUTP. To couple the aptamer to a DIII scaffold, succinimidyl 6-hydrazinonicotinoylacetonizone (SANH) can react with DIII scaffold lysine residues (lower panel). The bis-arylhydrazone bond between the two molecules is UV traceable at 354nM, and therefore the coupling ratio can be determined spectrophotometrically. After purification, the ability of all conjugated products to bind to the target can be assessed in vitro (cells) and then in vivo (xenografted mice).

Aptamer conjugation chemistry to scaffold DIII (shown as filled circles). The first reaction step is followed by a desalting step to remove unreacted SANH.

Bioconjugation of target-specific peptides or other proteins is achieved by using two heterobifunctional linkers. One is the synthesis of aromatic groups at the C-or N-terminus of peptides or proteinsHydrazine [ 6-hydrazinonicotinamide (HyNic)]. The other is an aromatic aldehyde [ 4-formylbenzamide (4FB) bonded to a random lysine (K) residue of the DIII protein]. The 4FB incorporation process is referred to as "modification" of DIII. After modification, the functionalized DIII and peptide molecules are desalted to remove excess linker and the biomolecules are exchanged into a coupling compatible buffer system. The two modified biomolecules are then mixed together and thermally stabilized between the two substances and can be incorporated into A_354nmCoupling occurs with bis-aryl hydrazone as determined spectrophotometrically. The peptide/DIII ratio was then calculated. The coupling reaction can be accomplished using commercially available reagents from SoluLink, Inc. (san Diego, Calif.).

Example 4.

Evaluation of anti-CEA peptide-DIIIb conjugates:

CEA-specific cyclic peptides (SDWVCEFIKSQWFCNVLASG, Kd 160nM) were commercially synthesized with a HyNic group at the C-terminus (SoluLink). The peptide precipitates when dissolved in aqueous solution. The lack of solubility in water immediately renders this peptide unsuitable for in vivo use. Prior to coupling, the HyNic modified peptide was dissolved in the organic solvent Dimethylformamide (DMF). Purified DIIIb was modified to incorporate a 4FB moiety at random lysine residues according to the protocol provided by SoluLink. After conjugation, the peptide/DIII ratio was determined by measuring a354nm, averaging 2 CEA-specific peptides per DIIIb molecule, and the conjugate was soluble in aqueous solution. Size exclusion chromatography was performed using a Superdex 200 column (GE healthcare, ciscativol, new jersey) for purification. Then use¹²⁴Purified anti-CEA peptide-DIIIb conjugates were labeled I and injected intravenously with 4 athymic nude mice loaded with LS174T (CEA positive) and C6(CEA negative) tumors. Mice were imaged with small animal PET/CT at 4, 20 and 27 hours, after which the mice were sacrificed, dissected and tissues/organs counted in a gamma well counter. The percentage injected dose per gram (% ID/g) calculated 27 hours after injection is shown in table 4 below.

PET/CT images (figure 10) demonstrate the ability of the peptide-DIIIb conjugate to target CEA positive tumors. High circulating activity was noted, indicating that the function of DIIIb to extend the circulating half-life of tumor targeting peptides was retained. However, the targeting moiety (peptide) is not able to bind the target efficiently, causing the peptide-DIIIb conjugate to dissociate and return to the circulation. Biodistribution data confirmed this (table 4) with relatively low uptake of LS174T tumor and high blood activity at 27 hours post injection.

Table 4.¹²⁴Biodistribution of I-labeled anti-CEA peptide-DIIIb conjugates in tumor-bearing mice (n ═ 4)

Tissue/organ	％ID/g	Standard deviation of
			Blood, blood-enriching agent and method for producing the same	3.00	0.32
Liver disease	1.42	0.08
			Spleen	1.04	0.16
Kidney (A)	3.66	0.66
			Lung (lung)	2.36	0.87
Muscle	0.30	0.03
			Stomach (stomach)	1.07	0.19
LS174T tumor	1.53	0.20
			C6 tumor	1.26	0.20
Cadaver	0.56	0.08

A tumor/muscle ratio of 5.1 was acceptable and comparable to antibody imaging. The tumor/blood and (CEA positive) tumor/(CEA negative) tumor ratios were relatively low (0.51 and 1.2, respectively), indicating high activity in blood and reduced tumor targeting. This observation again indicates that the conjugate remains in the blood long enough (DIIIb function) but that the peptide does not bind the target efficiently (CEA expressed by LS174T tumor). It is believed that peptides with high affinity (e.g., Kd < 10nM) are preferred for imaging of coupled DIII because affinity by itself cannot compensate for weak affinity.

Reference to the literature

Aina, o.h., Liu, r., Sutcliffe, j.l., Marik, j., Pan, c.x., and Lam, K.S. (2007) mol.pharm., 4, 631-.

Andersen, j.t., de Qian, j. and Sandlie, I. (2006) eur.j.immunol., 36, 3044-.

Barbas, a.s. and White, R.R. (2009) curr. opin. investig. drugs, 10, 572-578.Bebbington, c.r., Renner, g., Thomson, s., King, d., Abrams, d. and Yarranton, G.T. (1992) biotechnology (n y), 10, 169-.

Bembenek, s.d., Tounge, b.a., and Reynolds, C.H. (2009) Drug discov.today, 14, 278-.

Bos, o.j., Labro, j.f., Fischer, m.j., Wilting, j, and Janssen, L.H, (1989) j.biol.chem., 264, 953-.

Burger, a.m., Hartung, g., Stehle, g., Sinn, h., and Fiebig, H.H, (2001) int.j. cancer, 92, -.

Carmichael, j.a., Power, b.e., Garrett, t.p., Yazaki, p.j., Shively, j.e., raubishechek, a.a., Wu, a.m., and Hudson, P.J (2003) j.mol.biol., 326, 341-351.Chaudhury, c.s., Brooks, c.l., Carter, d.c., Robinson, j.m., and Anderson, c.l. (2006) Biochemistry, 45, 4983-.

Chaudhury, c., Mehnaz, s., Robinson, j.m., Hayton, w.l., Pearl, d.k., ropenian, d.c., and Anderson, c.l. (2003) j.exp.med., 197, 315-.

Chen, r., Li, l, and Weng, z. (2003) Proteins, 52, 80-87.

Curry, s., mantelkow, h., Brick, p., and Franks, N. (1998) nat. struct. biol., 5, 827-.

D' Argenio, D.Z. and Schumitzky, A. (1979) Compout.programs biomed., 9, 115-one 134.

Defrise, m., Kinahan, p.e., Townsend, d.w., Michel, c., Sibomana, m., and Newport, D.F, (1997) IEEE Trans med. imaging, 16, 145-.

Dugaiczyk, a., Law, s.w., and Dennison, O.E, (1982) proc.natl.acad.sci.usa, 79, 71-75.

Friedman, m., Nordberg, e., hodden-Guthenberg, i., Brismar, h., Adams, g.p., Nilsson, f.y., Carlsson, j. and Stahl, S. (2007) Protein eng.des.sel., 20, 189-.

Gentile, F., Amodeo, P., Febbraio, F., Picaro, F., Motta, A., Formisano, S., and Nucci, R. (2002) J.biol.chem., 277, 44050-.

Gronwall, C. and Stahl, S. (2009) J.Biotechnol., 140, 254-.

Kenanova, v., et al (2005) Cancer res., 65, 622-.

Kratz, F., Muller-Driver, R., Hofmann, I., Drevs, J., and Unger, C. (2000) J. Med. chem., 43, 1253-.

Loening, A.M. and Gambir, S.S. (2003) mol.imaging, 2, 131-.

Martin, w.l., West, a.p., jr., Gan, l., and Bjorkman, P.J, (2001) mol.cell, 7, 867-.

Melder, R.J., et al (2005) Cancer Immunol.Immunother, 54, 535-547.

Muller, d., Karle, a., Meissburger, b., Hofig, i., Stork, r., and Kontermann, R.E. (2007) j.biol.chem., 282, 12650-.

Nuttall, s.d. and Walsh, R.B. (2008) curr. opin. pharmacol., 8, 609-.

Olafsen, t., Kenanova, v.e., and Wu, A.M, (2006) nat. protocol, 1, 2048-.

Osborn, b.l., Olsen, h.s., nardell, b., Murray, j.h., Zhou, j.x., Garcia, a., Moody, g., Zaritskaya, l.s., and Sung, c. (2002) j.pharmacol.exp.ther., 303, 540-cup 548.

Saerens, d., Ghassabeh, g.h., and muydermans, S. (2008) curr. opin. pharmacol., 8, 600-.

Simonsen, s.m., Sando, l., Rosengren, k.j., Wang, c.k., Colgrave, m.l., Daly, n.l., and Craik, D.J, (2008) j.biol.chem., 283, 9805-.

Sugio, s., Kashima, a., Mochizuki, s., Noda, m., and Kobayashi, K. (1999) proteineeng, 12, 439-.

Venisnik, K.M., Olafsen, T., Gambir, S.S., and Wu, A.M, (2007) mol.imaging.biol., 9, 267-277.

Venisnik, K.M., Olafsen, T., Loening, A.M., Iyer, M., Gambir, S.S., and Wu, A.M, (2006) Protein Eng.Des.Sel., 19, 453-.

Wosikowski, k., Biedermann, e., ratetel, b., Breiter, n., Jank, p., Loser, r., Jansen, g., and Peters, G.J. (2003) clin.

Wu, a.m., Williams, l.e., Zieran, l., Padma, a., Sherman, m, Bebb, g.g., Odom-Maryon, t., Wong, j.y.c., Shively, j.e., and Raubitschek, A.A (1999) Tumor Targeting, 4, 47-58.

Wu, a.m., Williams, l.e., Zieran, l., Padma, q., Sherman, m.a., Bebb, g.g., Odom-Maryon, t., Wong, j.y.c., Shively, j.e., and Raubitschek, A.A (1999) turbine Targeting, 4.

Yazaki, p.j., Kassa, t., chenng, c.w., Crow, d.m., Sherman, m.a., Bading, j.r., Anderson, a.l., Colcher, d, and Raubitschek, a. (2008) nuclear.med.biol., 35, 151-.

Yazaki, p.j., Wu, a.m., Tsai, s.w., Williams, l.e., Ikler, d.n., Wong, j.y., Shively, j.e., and Raubitschek, A.A (2001) bioconjugateg.chem., 12, 220-.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (38)

1. A construct, comprising:

a) a protein scaffold, wherein the protein scaffold comprises domain III, domain IIIa or domain IIIb of human serum albumin or a polypeptide having substantial sequence identity to said domain III, domain IIIa or domain IIIb;

b) a targeting moiety covalently linked to the protein scaffold; and

c) a therapeutic moiety or an imaging moiety covalently linked to the protein scaffold.

2. The construct of claim 1, wherein the targeting moiety is a ligand that binds to a receptor of a target tissue or cell.

3. The construct of claim 1, wherein the targeting moiety is an antibody, or an immunologically active fragment thereof, that binds to a tumor-specific antigen.

4. The construct of claim 3, wherein the antibody is an immunologically active fragment of the antibody, a diabody, a triabody, or a minibody.

5. The construct of claim 1, wherein the targeting moiety is an aptamer.

6. The construct of claim 5, wherein the aptamer binds to a tumor specific antigen.

7. The construct of claim 2, wherein the ligand binds to a protein that is overexpressed in a target tissue or cell.

8. The construct of claim 2, wherein the target tissue or cell is a cancer.

9. The construct of claim 1, wherein at least one targeting, imaging, or therapeutic moiety is covalently bound to the scaffold via a non-peptide linker.

10. The construct of claim 1, wherein the substantial identity is 90%.

11. The construct of claim 1, wherein the substantial identity is 95%.

12. The construct of claim 1, wherein at least one of the targeting moiety, imaging moiety or therapeutic moiety is covalently bound to the scaffold via a heterobifunctional crosslinker, a homobifunctional crosslinker, a zero length crosslinker, a disulfide bond or a physiologically cleavable crosslinker.

13. The construct of claim 1, wherein the targeting moiety is covalently bound to the scaffold through a linker of 2-20 atoms in length.

14. The construct of claim 1, wherein the imaging moiety or the therapeutic moiety is bound to the scaffold via a linker of 2-20 atoms in length.

15. The construct of claim 1, wherein the construct has a molecular weight of less than 40 kda.

16. The construct of claim 1, wherein the construct has a molecular weight of less than 30 kda.

17. The construct of claim 1, wherein the construct has a molecular weight of less than 20 kda.

18. The construct of any one of claims 1 to 17, wherein domain III is wild-type or has a mutation at H535, H510 or H464.

19. The construct of claim 18, wherein the mutation is H535A, H510A, or H464A.

20. The construct of any one of claims 1 to 17, wherein the protein scaffold consists essentially of domain III, domain IIIa or domain IIIb.

21. The construct of any one of claims 1 to 17, with the proviso that the targeting moiety is not linked to the scaffold by a peptide bond.

22. The construct of any one of claims 1 to 17, with the proviso that the imaging and therapeutic moiety is not linked to the scaffold by a peptide bond.

23. The construct of any one of claims 1 to 17, wherein the construct comprises a therapeutic agent.

24. The construct of claim 23, wherein the therapeutic moiety is a drug.

25. The construct of any one of claims 1 to 17, wherein the therapeutic moiety is a therapeutic radionuclide, a cytotoxic drug, a cytokine, a chemotherapeutic agent, a radiosensitizer, or an enzyme.

26. The construct of claim 25, wherein the plurality of therapeutic moieties are covalently linked to the scaffold.

27. The construct of any one of claims 1 to 17, wherein the construct comprises an imaging agent.

28. The construct of claim 27, wherein the imaging agent is selected from the group consisting of: radionuclides, diamagnetic materials, fluorescent labels, chromogens, quantum dots, nanoparticles, and bioluminescent enzymes.

29. The construct of claim 27, wherein the plurality of imaging agents are covalently linked to the scaffold.

30. A method of detecting a disease or condition-associated biomolecule in a subject, the method comprising administering to a subject suspected of having or having the disease or condition the construct of claim 27, wherein a targeting moiety of the construct binds to the biomolecule and detecting an imaging agent of the construct.

31. The method of claim 30, wherein the presence or absence of said disease or disorder is diagnosed.

32. The method of claim 30, wherein the biomolecule is a tumor specific antigen and the disease or disorder is cancer.

33. The method of claim 30, wherein said biomolecule is a cell surface receptor or protein that is overexpressed or underexpressed in cells of a subject having said condition.

34. A method of treating a disease or condition associated with the presence of overexpression of a biomolecule in a tissue, the method comprising administering to a subject having the disease or condition a therapeutically effective amount of a composition according to claim

23. The construct, wherein a targeting moiety of the construct binds to the biomolecule and the therapeutic agent treats the disease or disorder.

35. The method of claim 34, wherein the targeting moiety binds a tumor-specific antigen of a cancer, and the disease or disorder is the cancer, and the therapeutic agent is a therapeutic radionuclide, a cytotoxic drug, a cytokine, or a chemotherapeutic agent.

36. A pharmaceutical or diagnostic composition comprising the construct of any one of claims 1-17 and a physiologically acceptable excipient or carrier.

37. Use of a construct according to any one of claims 1 to 17 in the manufacture of a medicament for the treatment of a disease or disorder.

38. Use of a construct according to any one of claims 1 to 17 in the manufacture of a diagnostic agent for the detection of a disease or condition.