# Patent

## Title
Cysteine-containing peptide tag for site-specific conjugation of proteins

## Abstract

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-in-Part application of U.S. Ser. No. 09/872,712 filed Jun. 1, 2001, now abandoned, which claims the benefit of U.S. Provisional Application 60/209,660 filed Jun. 6, 2000, both of which are incorporated by reference herein in their entireties.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made in part with government support under grant numbers 1 R43 HL61143-01; 1-R43-CA113080-01; R43AI054060-01; and 1 R43 GM072170-01 from the National Institutes of Health, and grant number DE-FG-02-02ER83520 from the Department of Energy. The government has certain rights in this invention.


BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention is directed to nucleic acid and protein sequences that encode a cysteine-containing tag and a targeting protein. The present invention is also directed to a biological conjugate comprising proteins having the above protein sequences and a binding moiety covalently bound to the cysteine-containing tag in the protein. The present invention is also directed to pharmaceutical compositions containing the biological conjugates in combination with selected therapeutic, diagnostic, or research entities, and methods of administering the pharmaceutical compositions to a patient to achieve targeted delivery of the therapeutic, diagnostic, or research entities in the patient. 2. Description of the Related Art.
Conjugation of recombinant proteins to various entities is used in several areas. One area is targeted delivery of therapeutic, diagnostic, and research agents to targeted cells in the patient in order to improve their efficacy and to minimize potentially adverse side effects. In this area either therapeutic, diagnostic, and research agents, or their carriers are chemically conjugated to recombinant targeting proteins that can selectively bind to targeted cells (reviewed by Dubowchik & Walker, 1999). The resulting conjugates are structurally and functionally heterogeneous because they are formed randomly via chemical reactions with few of several available chemical groups, usually ε-amino groups of lysine residues, in the targeting protein. Since random conjugation does not discriminate between functionally important and dispensable amino acid residues in the targeting protein, the procedure should be custom-developed and optimized on a case-by-case basis in order to increase the proportion of functionally active proteins.
Another area is derivatizing artificial surfaces and/or bulk compositions of biomedical devices or tissue scaffolds with proteins that target certain components of intra-organism environment in order to improve surface compatibility with the environment and to modulate the desired features, such as affinity or rejection of certain intra-organism components. In this area recombinant proteins are covalently grafted on the material through random chemical conjugation, usually via ε-amino groups of lysine residues, that involves both functionally important and dispensable amino acid residues in the proteins, resulting in heterogeneous products with unknown fraction of functionally active proteins.
Yet another area with similar problems is construction of various biosensors or other functional devices with protein-derivatized surfaces that convert the results of interactions between the “working” protein and the targeted components of the environment into a detectable output, including but not limited to a detectable signal or the products of enzymatic activity of immobilized proteins. In this area recombinant proteins are also chemically conjugated to artificial surfaces of these devices usually via ε-amino groups of lysine residues, yielding heterogeneous surfaces with unknown fraction of functionally active proteins.
Several methods for chemical conjugation of proteins to artificial surfaces have been developed (see, for example U.S. Pat. No. 5,492,840 to Malmqvuist; U.S. Pat. No. 5,853,744 to Mooradian et al.; U.S. Pat. No. 6,033,719 to Keogh, Mann et al. (2001); Kuhl & Griffith-Cima, (1996); Bentz et al. (1998). These methods were developed on a case-by-case basis in order to minimize damage to the protein and to increase the homogeneity of the surface.
These problems are well recognized, and over the years several approaches have been developed for introduction into recombinant proteins of unique cysteine residues for site-specific conjugation of various entities. This strategy is based on observation that intrinsic cysteine residues in proteins are usually involved in intramolecular or inter-subunit disulfide bonds and are not readily available for chemical conjugation. In theory, introduction of a unique cysteine residue that does not affect formation of intrinsic disulfide bonds and does not affect functional activity of the recombinant protein can provide a thiol group available for site-specific conjugation via chemistries known in art. For example, several groups reported introduction of cysteines into recombinant single-chain Fv antibody fragments (scFv), usually at or near C-termini, in order to use these cysteine residues for formation of diabodies and/or for site-specific conjugation to various entities (Adams et al., 1993; Kipriyanov et al., 1994; Wang et al., 1997; Marti et al., 2001; Gupta et al., 2001; Xu et al., 2002; Li et al., 2002; Renard et al., 2002; Albrecht et al., 2004). However, even for scFv, the presence of unpaired cysteine at or near the C-terminus significantly affects protein yield, solubility and functional activity (Schmiedl et al., 2000). Futami et al. (2000) introduced cysteine residues near the N- and C-termini of into human RNase I which resulted in a stabilized RNase I. However, yield and enzymatic activity of the product were significantly reduced. Moreover, this mutant RNase I or its fragments were not used in other products.
Another method for site directed modification of proteins is intein-mediated ligation of various entities to the C-terminus of the protein (see, for example Evans et al., 1999; Tolbert and Wong 2000; Macmillan et al., 2000; Mukhopadhyay et al., 2001; Hofmann, and Muir, 2002; Lovrinovic et al., 2003; Wood et al., 2004). However application of this method require proper folding of the protein fused to a large intein domain and the ability to withstand fairly harsh reducing conditions during intein-mediated ligation. Furthermore, in both approaches discussed above, conjugation to available cysteine residue is limited to entities that do not interfere with activities of the protein despite their close proximity to the body of the protein.
Thus, in the area of protein-based targeted delivery of therapeutic, diagnostic, and research compounds, as well as in the area of construction of various devices and scaffolds with protein-derivatized surfaces, what is needed in the art are compositions and general methods that (1) allow for site-specific conjugation of recombinant proteins to various entities in order to produce more homogeneous products in ways that minimize interference with functional activities of said proteins; (2) readily convert various recombinant proteins of interest into a format suitable for site-specific conjugation; (3) can be utilized with a wide variety of entities to which a recombinant protein of interest need to be conjugated; and (4) do not result in immunogenic or toxicity problems when introduced into humans. The present invention is believed to be an answer to these objectives.
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to an isolated polypeptide, consisting of the sequence of SEQ ID NO:2.
In another aspect, the present invention is directed to an isolated nucleic acid consisting of a nucleic acid sequence that encodes the polypeptide sequence of SEQ ID NO:2.
In another aspect, the present invention is directed to an isolated polypeptide, comprising the sequence of SEQ ID NO:4.
In another aspect, the present invention is directed to an isolated nucleic acid comprising a sequence that encodes the polypeptide sequence of SEQ ID NO:4.
In another aspect, the present invention is directed to an isolated polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein.
In another aspect, the present invention is directed to an isolated nucleic acid encoding the polypeptide sequence of SEQ ID NO:6, 8, 10, 12, or 14.
In another aspect, the present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety.
In another aspect, the present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein bound covalently to the targeting moiety, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein.
In another aspect, the present invention is directed to a pharmaceucial composition for selectively delivering selected entities to a target in a patient, comprising a pharmaceutically acceptable carrier; and one or another of the above biological conjugates.
In another aspect, the present invention is directed to a method of selectively delivering entities to a target in a patient, comprising the steps of: (a) administering to a patient the above pharmaceutical composition; and (b) permitting the biological conjugate to contact the target to deliver the entity to the target in the patient.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a representation of amino acid and nucleic acid sequences of C4-tag genetically fused to multiple cloning site region via a G4S linker (Panel A), and a schematic representation of a plasmid for bacterial expression of fusion recombinant proteins fused to C4-tag via a G4S linker (Panel B);
FIG. 2 is a schematic representation of site-specific conjugation between C4-tagged fusion recombinant protein and a complimentary adapter protein, named HuS(C118) (Panel A), a schematic representation of a family of adapter proteins based on human RNase I capable of site-specific conjugation to cysteine residue in C4-tag genetically fused to recombinant protein (Panel B), a demonstration of spontaneous conjugation via a disulfide bond between C4-VEGF and HuS(C118) leading to appearance of DTT-sensitive new protein bands in samples named HuS-C4-VEGF on the SDS-PAGE gel (Panel C), and a demonstration that ribonuclease activity is reconstituted upon chemical conjugate formation, but not upon physical mixing of adapter protein with C4-tagged recombinant fusion protein (Panel D).
FIG. 3 illustrates that functional activities of several C4-tagged recombinant fusion proteins and C4-tagged recombinant fusion proteins conjugated to complimentary adapter protein are comparable to that of parental proteins. Panel A illustrates that in VEGFR-2 tyrosine phosphorylation assay in 293/KDR cells functional activities of C4-tagged vascular endothelial growth factor, (C4-VEGF), C4-tagged single-chain vascular endothelial growth factor (C4-scVEGF), and HuS(C 118)-C4-VEGF conjugate (HuS-C4-VEGF) are comparable with that of VEGF that does not contain C4-tag. Panel B illustrates that functional activities of C4-tagged annexin V (C4-annexin) and HUS(C118)-C4-annexin V conjugates (HuS-C4-annexin) are comparable with that of annexin V in an erythrocyte binding assay. Panel C illustrates that ability of a C4-tagged catalytically inactive fragment of anthrax lethal factor, C4-LFn, and the corresponding HuS(C118)-C4-LFn conjugate (HuS-C4-LFn) are comparable to that of LFn in protection of RAW cells from cytotoxic activity of anthrax toxin LF/PA.
FIG. 4 illustrates site-specific conjugation of HYNIC-maleimide (5-maleimido-2-hydraziniumpyridine hydrochloride), a chelator for 99mTc, to C4-VEGF and C4-scVEGF fusion proteins and functional activity of such conjugates. Panel A illustrates that functional activities of HYNIC-C4-VEGF and HYNIC-C4-scVEGF conjugates are comparable to that of parental C4-VEGF in VEGFR-2 tyrosine autophosphorylation assay in 293/KDR cells. Panel B illustrates that HYNIC-C4-VEGF and HYNIC-C4-scVEGF conjugates are comparable with parental C4-VEGF in their abilities to protect 293/KDR cells from cytotoxicicity of toxin-VEGF fusion protein.
FIG. 5 illustrates site-specific conjugation of 20 kDa or 40 kDa maleimide-polyethyleneglycol (PEG20, and PEG40, correspondingly) to C4-VEGF and functional activity of the PEG-C4-VEGF conjugate. Panel A illustrates SDS-PAGE analysis of PEG-C4-VEGF conjugate. Panel B illustrates that functional activity of PEG-C4-VEGF conjugate is comparable to that of parental C4-VEGF in VEGFR-2 tyrosine autophosphorylation assay in 293/KDR cells.
FIG. 6 illustrates the use of C4-tag and a complimentary adapter protein HuS(C88, C118) for construction of VEGF-driven conjugates (Cy5.5-Hus-C4-VEGF) containing cyanine dye Cy5.5 for targeted near-infrared fluorescent imaging in vivo. Panel A is a flow-chart for construction and characterization of Cy5.5-Hus-C4-VEGF conjugate. Panel B illustrates that functional activity of Cy5.5-Hus-C4-VEGF conjugate is comparable to that of parental C4-VEGF in VEGFR-2 tyrosine autophosphorylation assay, while site-specific (Cy5.5-C4-VEGF) or random conjugation (Cy5.5-VEGF) of a similar amount of Cy5.5 per C4-tagged VEGF decreases activity of VEGF. Panel C illustrates that Cy5.5-Hus-C4-VEGF conjugate is comparable to that of parental C4-VEGF in their ability to protect 293/KDR cells from cytotoxicity of toxin-VEGF fusion protein, while site-specific (Cy5.5-C4-VEGF) or random conjugation (Cy5.5-VEGF) of a similar amount of Cy5.5 per C4-tagged VEGF decreases VEGF activity. Panel D illustrates the use of Cy5.5-Hus-C4-VEGF conjugate for in vivo imaging of tumor vasculature.
FIG. 7 illustrates the use of C4-tag and a complimentary adapter protein HuS(C118) for construction of VEGF-driven conjugates (Lip/Hus-C4-VEGF) containing doxorubicin-loaded liposomes (“DOXIL”) for targeted drug delivery. Panel A is flow-chart for construction and characterization of Lip/Hus-C4-VEGF conjugate. Panel B illustrates that functional activity of Lip/Hus-C4-VEGF conjugate is comparable with that of parental C4-VEGF in VEGFR-2 tyrosine autophosphorylation assay. Panel C illustrates that VEGF-targeted doxorubicin-loaded liposomes (Lip/HuS-C4-VEGF) are toxic to VEGFR-2 expressing cells in a concentration range where untargeted doxorubicin-loaded liposomes are ineffective underlying receptor-mediated mechanism of toxicity of Lip/Hus-C4-VEGF conjugate. Panel D illustrates that VEGF protects 293/KDR cells from cytotoxic activity of Lip/Hus-C4-VEGF conjugate.
FIG. 8 illustrates the use of C4-scVEGF for construction of VEGF-driven conjugates (Lip/C4-scVEGF) containing doxorubicin-loaded liposomes (“DOXIL”) for targeted drug delivery. Panel A is a flow-chart for construction and characterization of Lip/C4-scVEGF conjugate. Panel B illustrates that in VEGFR-2 tyrosine autophosphorylation assay functional activity of Lip/C4-scVEGF conjugate is comparable with that of parental C4-VEGF. Panel C illustrates that Lip/C4-scVEGF are toxic to VEGFR-2 expressing cells in a concentration range where untargeted doxorubicin-loaded liposomes are ineffective underlying receptor-mediated mechanism of toxicity of Lip/C4-scVEGF conjugate. Panel D illustrates that VEGF protects 293/KDR cells from cytotoxic activity of Lip/C4-scVEGF conjugate.



DETAILED DESCRIPTION OF THE INVENTION
As indicated above, the present invention comprises compositions and methods useful for site-specific conjugation of recombinant fusion proteins to various entities via a single cysteine residue present in a peptide tag engineered into the protein. Current conjugation methods known in the art rely mostly on random cross-linking of various entities to amino acid residues, such as, for example, lysine or tyrosine, that are abundant in the protein. Less abundant cysteine residues are usually involved in intramolecular disulfide bonds essential for the functional activity of the protein and therefore not available for conjugation. As a result, even when conjugation involves only one amino acid residue per protein, the final product contains a mixture of proteins modified at different positions and therefore heterogeneous in their activity, pharmacokinetics, pharmacodynamic, and tissue distribution characteristics. Furthermore, conjugation to amino acid residues in the protein is always limited by the harm it may inflict upon the functional activity of the proteins. As a result, conjugation procedures have to be custom-developed on a case-by-case basis. However, customized conjugation does not allow a standardized approach to rapid adaptation of different proteins for similar purposes, for example for a targeted delivery of the same imaging reagent, or surface derivatization of the same device.
To overcome these and other obstacles, the present invention discloses compositions and methods useful for site-specific conjugation of recombinant proteins to various entities. The method is based on conversion of a protein of interest into a fusion protein that comprises a cysteine-containing peptide tag, named C4, fused via a linker peptide to an N-terminus or a C-terminus of the protein of interest. By definition, in order to be useful, such fusion proteins should retain its functional activities, and a cysteine residue in the peptide tag should be available for site-specific conjugation with various entities, including complimentary adapter proteins, capable of forming a covalent disulfide bond with tag's cysteine. Various chemistries for conjugation to a cysteine thiol group are well-known in art. For example, entities derivatized with maleimide groups, or vinylsulfone groups, or with chemically activated thiol groups can be conjugated to a thiol group in C4-tag. On the other hand, thiol group in the C4-tag can be modified with formation of an activated disulfide bond that might react with available thiol groups on various entities, or it can be modified with bifunctional reagents for conjugation to entities that can react with the second functional group.
Thus, it has now been discovered that a cysteine-containing peptide fusion C4-tag can confer upon a protein fused to the tag the ability to be chemically conjugated to various chemical components. The cysteine residue of the C4 tag is used to form a covalent bond to the chemical components, thus providing a strong, stable linkage. Through the cysteine residue of C4, stable bonds may be formed between the C4 tagged protein and a wide variety of entities, including drugs, drug carriers, contrast agents, carriers for contrast agents, radionuclides, carriers for radionuclides, various nano- and microparticles, including liposomes, quantum dots, small and ultra-small paramagnetic particles, other proteins or protein fragments, nucleic acids, various protein-modifying molecules, including but not limited to polyethyleneglycol of various molecular weights, as well as surfaces of various devices, such as biomedical devices, biosensors, or artificial tissue scaffolds. In one embodiment, a complimentary adapter protein is covalently bonded to the C4 tagged protein through a disulfide bond. The adapter protein can be used a platform for conjugation to various entities described above. The present invention discloses that C4-tagged proteins retain protein functional activity and can be site-specifically conjugated to various entities without loss of functional activity either in vitro or in vivo.
The formation of a covalent bond offers several advantages for the present invention. First, the entity covalently bound to C4-tag is removed from the functional parts of the protein. Furthermore, a complimentary adapter protein allows further distancing of bound entities from the functional parts of the protein. Second, the covalent bond is strong such that the materials bound to the C4 tagged protein do not easily dissociate in biological fluids or in solutions. Third, the chemistry and conditions to form the covalent bond known and can be easily reproduced. Fourth, under certain conditions known to those of skill in the art, it is possible to destroy the covalent linkage and permit the components to dissociate. For example, if a disulfide bond is formed, appropriate conditions can be selected to reduce this bond and permit the C4-tagged protein to dissociate. This feature of the invention offers advantages in that, for example, that derivatized surfaces can be regenerated after a predetermined length of time.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs.
As used herein, “C4”, refers to a mutant N-terminal 15 amino acid long fragment of human RNase I with the following nucleic acid and amino acid sequences:








(SEQ ID NO: 1)









AAAGAATCTTGCGCTAAAAAATTTCAACGTCAACACATGGACTCT



 








(SEQ ID NO: 2)









Lys-Glu-Ser-Cys-Ala-Lys-Lys-Phe-Gln-Arg-Gln-His-



 


Met-Asp-Ser






Although minimally altered human peptides are not expected to be immunogenic in humans and therefore are preferred embodiments of cysteine-containing tags, it is understood that in some applications immunogenicity is not an issue, and therefore these peptides may be used as modified by amino acid substitutions, amino acid deletions, amino acid insertions, and amino acid additions, including placing of cysteine residue in different positions in these peptides or using corresponding fragments from non-human ribonucleases.
As used herein, “targeting protein” refers to a protein that can selectively interact with cellular receptors or other cell surface proteins, or can selectively interact with certain components of the environment, either free or bound to a surface.
As used herein, the term “linker sequence” refers to an innocuous length of nucleic acid or protein that joins two other sections of nucleic acid or protein.
The terms “mutant” and “mutated” refer to nucleic acid or protein sequences which are not found in nature. The term “truncated” refers to nucleic acid or protein sequences that are shorter than those found in nature.
As defined herein, “biological conjugate” refers to a complex between a targeting moiety and a binding moiety and stabilized by a covalent bond. “Targeting moiety” refers to a protein having a polypeptide comprising C4 and a targeting protein. “Binding moiety” refers to any substance or surface that can be covalently bound to the targeting moiety. The term “functional group” refers to natural or non-natural chemical groups that interact chemically with a thiol group. A natural functional group refers to a chemical group that is found naturally in the binding moiety that can interact chemically with a thiol group. A non-natural functional group refers to a group that is artificially added to the binding moiety (e.g., addition of a maleimide group or vinylsulfone group to polyethylene glycol) such that the artificial group is chemically reactive with the thiol group of the binding moiety.
“Adapter protein” refers to a protein that is complimentary (e.g., part of a binding pair) and can interact with the targeting moiety to form a specific disulfide bond between it and the targeting moiety. “Fusion protein” refers to a recombinant protein that contains two or more polypeptide fragments that are encoded by DNA sequences that have been combined with the methods of recombinant DNA technology in a form that allows expression of the fusion protein in suitable hosts.
As used herein, “carrier” refers to natural or synthetic molecules or aggregates thereof which can be associated covalently or non-covalently with therapeutic, diagnostic, or research compounds. Such carriers include, but are not limited to chelators, natural or synthetic polymers including dendrimers, co-polymers, derivatized polymers, liposomes, various viral and bacteriophage particles, various natural and manufactured nano- and microparticles, and beads.
As used herein, “scVEGF” refers to a single chain vascular endothelial growth factor that comprises two 3 to 112 amino acid residue fragments of the VEGF121 isoform connected head-to-tail into a single-chain protein via alanine residue and has the following protein and nucleic acid sequences:








(SEQ ID NO:4)









   NH2-Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr



 


Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu


 


Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu


 


Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln


 


His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala


 


Arg Ala Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln


 


Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr


 


Ile Phe Lys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu


 


Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His


 


Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Aia Arg-


 


COOH


 








(SEQ ID NO:3)









     ATGGCAGAAGGAGGAGGGCAGAATCATCACGAAGTGGTGAAGTTCATGGAT



 


GTCTATCAGCGCAGCTACTGCCATCCAATCGAGACCCTGGTGGACATCTTCCAGGAG


 


TACCCTGATGAGATCGAGTACATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGC


 


GGGGGCTGCTGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACAT


 


CACCATGCAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGA


 


GCTTCCTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAAGAGCC


 


ATGGCAGAAGGAGGAGGGCAGAATCATCACGAAGTGGTGAAGTTCATGGATGTCTA


 


TCAGCGCAGCTACTGCCATCCAATCGAGACCCTGGTGGACATCTTCCAGGAGTACCC


 


TGATGAGATCGAGTACATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGCGGGGG


 


CTGCTGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCACCA


 


TGCAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGAGCTTC


 


CTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAAGA TGA






Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NO:4 is embraced by the nucleic acids of the present invention.
In yet another embodiment, the targeting moiety has the polypeptide sequence shown in SEQ ID NO: 14. This polypeptide sequence, which is a genetic fusion of C4 and a single chain VEGF (scVEGF) having the polypeptide sequence shown in SEQ ID NO:4, is encoded by the nucleic acid sequence shown in SEQ ID NO:13. Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NO:14 is embraced by the nucleic acids of the present invention. Although scVEGF contains 16 cysteine residues per single-chain molecule, C4-tagged molecules are refolded into functionally active conformation, whereby the cysteine residue in C4-tag can be conjugated to various entities yielding conjugates with functional activities comparable to that of unmodified VEGF.
As indicated above, in one embodiment, the present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety; wherein the biological conjugate has a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is also directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein bound covalently to the targeting moiety, the adapter protein having a thiol group; wherein the biological conjugate has a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. Each of these components are discussed in more detail below.
The targeting moiety is preferably a protein having a polypeptide comprising the C4 peptide and a targeting protein. As indicated above, the C4 peptide portion of the targeting moiety is a mutant N-terminal 15 amino acid long fragment of human RNase I wherein arginine at position 4 has been substituted with cysteine. This particular peptide offers several advantages in the present invention. The human origin decreases the likelihood of inducing a strong immune response in a human host. Furthermore, an N-terminal fragment of human RNase I is capable of forming an α-helix that may protect it from forming disulfide bonds with other cysteine residues in the fusion protein during refolding and purification of the protein. Finally, enzymatically inactive wild type N-terminal and C-terminal fragments of human RNase I spontaneously form enzymatically active non-covalent complexes, a phenomenon that is exploited in the present invention for developing of complimentary adapter proteins, capable of forming disulfide bond with C4 residue in C4-tag. The mutant N-terminal 15 amino acid long fragment of human RNase I has the following nucleic acid and amino acid sequences:








(SEQ ID NO:1)









AAAGAATCTTGCGCTAAAAAATTTCAACGTCAACACATGGACTCT



 








(SEQ ID NO:2)









Lys-Glu-Ser-Cys-Ala-Lys-Lys-Phe-Gln-Arg-Gln-His-



 


Met-Asp-Ser






The targeting protein portion of the targeting moiety is any protein that can selectively bind to cellular receptors or other cell surface proteins or selectively interact with certain components of the environment, and is genetically fused to the C4 peptide. In preferred embodiments, the targeting protein may be human vascular endothelial growth factor (VEGF), or a mutated or truncated form thereof such as VEGF110, human annexin V, or a mutated or truncated form thereof, a catalytically inactive fragment of anthrax lethal vector, or a mutated or truncated form thereof, or a single chain VEGF derivative, or a mutated or truncated form thereof.
In one embodiment, the targeting moiety has the polypeptide sequences shown in SEQ ID NOS: 6 or 8. These polypeptide sequences, which are genetic fusions of C4 and VEGF121 or VEGF110, respectively, are coded by the nucleic acid sequences shown in SEQ ID NOS:5 and 7, respectively. Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NOS:6 or 8 are embraced by the nucleic acids of the present invention. In addition, although selected isoforms of VEGF (VEGF121) contains 18 cysteine residues per dimeric molecule, tagged molecules are refolded into functionally active conformation, whereby cysteine residue in C4-tag can be conjugated to various entities yielding conjugates with functional activities comparable to that of unmodified VEGF.
Vascular endothelial growth factor (VEGF) controls growth of endothelial cells via interaction with several receptors, among which KDR/flk-1 (VEGFR-2) receptor expression is limited mostly to endothelial cells. In adult organisms the growth of endothelial cells (angiogenesis) occurs, with the exception of corpus luteum development, only in various pathological conditions. Thus, KDR/flk-1 (VEGFR-2) receptor-mediated delivery of therapeutic, diagnostic, contrast, and research entities site-specifically linked to VEGF or scVEGF, or linked to VEGF or scVEGF via a complimentary adapter protein, may be useful in therapies for various pathologies. On the other hand, long-circulating, or slow-releasable from a suitable matrix, site-specifically PEGylated VEGF or scVEGF, as well as VEGF or scVEGF conjugated to the surfaces of biomedical devices, such as stents, or tissue scaffolds, might be useful for promotion of angiogenesis in ischemic situations.
In another embodiment, the targeting moiety has the polypeptide sequence shown in SEQ ID NO:10. This polypeptide sequence, which is a genetic fusion of C4 and annexin V, is encoded by the nucleic acid sequence shown in SEQ ID NO:9. Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NO:10 is embraced by the nucleic acids of the present invention. Although annexin V contains a single cysteine residue, tagged molecules are refolded into functionally active conformation, whereby the cysteine residue in C4-tag can be conjugated to various entities yielding conjugates with functional activities comparable to that of unmodified annexin V. Annexin V interacts with phosphatidylserine exposed on the surface of apoptotic cells, and is used as an early marker of apoptotic process. Thus, phosphatidylserine-mediated delivery of therapeutic, diagnostic, or research entities site-specifically linked to annexin V might be useful for inhibition or promotion of apoptosis.
In another embodiment, the targeting moiety has the polypeptide sequence shown in SEQ ID NO:12. This polypeptide sequence, which is a genetic fusion of C4 and a catalytically inactive fragment of anthrax lethal factor, known as LFn, is encoded by the nucleic acid sequence shown in SEQ ID NO:11. Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NO:12 is embraced by the nucleic acids of the present invention. Although LFn contains no cysteine residues, tagged molecules are refolded into functionally active conformation, whereby the cysteine residue in C4-tag can be conjugated to various entities yielding conjugates with functional activities comparable to that of unmodified LFn. Catalytically inactive fragment of anthrax lethal factor, LFn, pairs with another anthrax protein, named protective antigen (PA) that interacts with the same cellular receptors as the combination of catalytically active lethal factor/protective antigen (LF/PA). Thus, PA-mediated delivery of therapeutic, diagnostic, or research entities site-specifically linked to LFn might be useful for mapping sites with receptors for PA, or delivery to cells compounds that might interfere with cytotoxic activity of lethal factor.
In yet another embodiment, the targeting moiety has the polypeptide sequence shown in SEQ ID NO:14. This polypeptide sequence, which is a genetic fusion of C4 and a single chain VEGF (scVEGF) having the polypeptide sequence shown in SEQ ID NO:4, is encoded by the nucleic acid sequence shown in SEQ ID NO:13. Due to the redundancy of the genetic code, however, any nucleic acids that code for SEQ ID NO:14 is embraced by the nucleic acids of the present invention. As indicated above, scVEGF comprises two 3 to 112 amino acid residue fragments of the VEGF121 isoform connected head-to-tail into a single-chain protein via alanine residue. Although scVEGF contains 16 cysteine residues per single-chain molecule, tagged molecules are refolded into functionally active conformation, whereby the cysteine residue in C4-tag can be conjugated to various entities yielding conjugates with functional activities comparable to that of unmodified dimeric VEGF.
With reference to the above targeting moiety, a linker sequence may be positioned between the C4 peptide and the sequence of the targeting protein. Linker sequences, such as Gly4Ser or (Gly4Ser)3 linkers, are engineered in the commercially available vectors for bacterial expression of recombinant proteins and can be readily engineered into vectors for expression of recombinant proteins in other hosts, including, but not limited to mammalian cells, insect cells, yeast cells, and transgenic organisms. Linkers serve to provide some useful distance between the C4 peptide and the targeting protein. Although in the presented embodiments C4-tag is positioned at the N-terminus of targeting protein, one skilled in the art would appreciate that the tag may be placed at the C-terminus of the targeting protein, or inside the functionally dispensable area of targeting protein, for example between two functional domains of single-chain antibody, using commonly known methods of genetic engineering.
FIG. 1, Panel A, shows amino acid and nucleic acid sequences of C4-tag genetically fused to a multiple cloning site region found in the typical expression plasmid via a G4S linker. As shown in Panel A of FIG. 1, the full nucleic acid sequence includes control and transcription elements (T7 promotor, lac operatior, ribosome binding site, and the like) as well as the nucleic acid sequence of the C4 tag. A linker sequence separates the C4 sequence from a multiple cloning site, which may be used to introduce nucleic acids of interest that will function as the targeting protein. A T7 termination site completes the full length nucleic acid. FIG. 1, Panel B, shows a schematic representation of a plasmid for bacterial expression of fusion recombinant proteins fused to C4-tag via a G4S linker.
The targeting moiety of biological conjugate may be cytokines, chemokines, growth factors, antibodies and their fragments, enzymes, and combinations of thereof that may be useful in various biomedical or industrial applications. The binding moiety portion of the biological conjugate may be any substance or surface that can be covalently bound to the C4-thiol group of targeting moiety or to functional group of adapter protein in the adapter/targeting moiety conjugate. Examples of useful binding moieties include, but are not limited to, drugs, radionuclide chelators, polyethylene glycol, dyes, lipids, liposomes, and selected surfaces.
Useful radionuclide chelators include compounds such as 5-maleimido-2-hydraziniumpyridine hydrochloride (HYNIC) for loading with imaging or therapeutic radionuclide. Polyethylene glycol is useful for slowing blood clearance of protein, and may be used in a derivatized (e.g., modified with maleimide or vinylsulfone) or underivatized forms. Useful dyes include, for example, cyanine dye Cy5.5 for near-infrared fluorescent imaging. Surfaces that may bind to the C4-thiol group of targeting moiety or to functional group of adapter protein in the adapter/targeting moiety conjugate include surfaces of a nano- or microparticle, the surface of a dendrimer, surfaces of tissue culture scaffolds, biomedical devices, and biosensors. It is understood that when a binding entity include surface, it may be used as such, or may have other chemical groups deposited on the surface by methods known in art. These chemical groups might be further used for modification with bifunctional reagents that allow conjugation to C4-tag or to adapter protein via methods known in art.
In one embodiment of the present invention, the biological conjugate has a covalent bond between the thiol group of the C4 peptide (SEQ ID NO:2), and a naturally occurring functional group in the binding moiety. Thus, it is contemplated that the thiol group of the C4 peptide reacts chemically directly with a reactive group in the binding moiety such that a stable covalent bond is formed. In alternative embodiments, the reactive group that reacts with the thiol group of the C4 peptide may be introduced artificially, for example by using bifunctional crosslinking agents known in art. For example, a maleimide group can be introduced into polyethylene glycol or a lipid and used for reaction with the thiol group of the C4 peptide.
In an alternative embodiment, the biological conjugate of the present invention includes a binding moiety that comprises adapter protein bound covalently to the targeting moiety. In general, the adapter protein is a mutant human RNase I, or a fragment thereof, that includes cysteine at position 118 (Cys118). Examples of adapter proteins useful in the present invention include proteins having polypeptide sequences shown in SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22, which are described in more detail below.
Four particularly preferred embodiments of adapter protein capable of forming a disulfide bond with C4 residue in C4-tag are shown in FIG. 2 where Panel A provides a schematic representation of site-specific conjugation between C4-tagged fusion recombinant protein and a complimentary adapter protein. FIG. 2, Panel B shows a schematic representation of a family of adapter proteins based on T24N, V118C mutant human RNase I. The first embodiment is a fragment of T24N, V118C mutant human RNase I, named HuS(C118) (SEQ ID NOS:15 and 16). Amino groups of HuS(C118) can be used for conjugation to various entities using methods known in art prior to site-specific conjugation of HuS(C118) to C4-tagged proteins. The second embodiment is a fragment of T24N, V118C mutant human RNase I containing the N88C substitution, named HuS(C88, C118) (SEQ ID NOS:17 and 18). C88 thiol group in HuS(C88, C118) can be used for conjugation to various entities using methods known in art after site-specific conjugation of HuS(C88, C118) to C4-tagged proteins. The third and fourth embodiments are based on a chimeric BH-RNase that contains a 1-29-aa fragment of bovine RNase A that differs from a corresponding fragment of human Rnase I in several positions and a 30-127 aa fragment of human RNase I (Gaynutdinov et al., 2003). The third embodiment is the V118C mutant of BH-RNase containing the F8A and Q11P, substitutions, named BHR(A8, P11, C118) (SEQ ID NOS:19 and 20) which does not require removal of 20 N-terminal amino acid residues prior to site-specific conjugation to C4-tagged proteins. Amino groups of BHR(A8, P11, C118) can be derivatized with various entities using methods known in art prior to site-specific conjugation of HuR(A8, P11, C118) to C4-tagged proteins. The fourth embodiment is the V118C mutant BH-RNase containing A8, P11, and C88, named BHR(A8, P11,C88, C118) (SEQ ID NOS:21 and 22) which does not require removal of 20 N-terminal amino acid residues prior to site-specific conjugation to C4-tagged proteins. The C88 thiol group in BHR(A8, P11,C88, C118) can be used for conjugation with various entities using methods known in art after site-specific conjugation of BHR(A8, P11,C88, C118) to C4-tagged proteins. Using methods known in art it will be easy to construct other adapter proteins based on human RNase I capable of site-specific conjugation to C4-tagged proteins and providing convenient platforms for derivatization with various entities. One of skill in the art would appreciate that other adapter proteins based on human RNase I may be used in addition to those explicitly described herein. For example, one of skill in the art would appreciate that adapter proteins based on other ribonucleases, for example bovine RNase A, may be constructed as long as these adapter proteins retain the ability to form conjugates with C4-tag, or similar tags based on N-terminal fragments of other ribonucleases.
Panel C in FIG. 2 provides evidence of C4-VEGF and HuS(C118) conjugation via a disulfide bond leading to the appearance of DTT-sensitive new protein bands in samples named HuS-C4-VEGF on the SDS-PAGE gel corresponding to conjugation of one or two adapter HuS(C118) to C4-tags in a dimeric molecule of C4-VEGF. Panel D provides evidence that ribonuclease activity is reconstituted upon chemical conjugate formation, but not upon physical mixing of HuS(C118) adapter protein with C4-tagged recombinant fusion protein (Panel D).
The adapter protein may be derivatized with a host of functional materials to achieve a desired result. For example, in one embodiment, the adapter protein may be derivatized with a dye, such as Cy5.5 dye for optical imaging, or with a lipid for associating the adapter protein with a liposome that acts as a carrier for therapeutic, diagnostic, or research compounds. In one embodiment, the liposomes may carry or be loaded with doxorubicin (“DOXIL”).
The biological conjugates may be combined with pharmaceutically acceptable carriers to produce pharmaceutical compositions for selectively delivering therapeutic, research, or diagnostic compounds to a target in a patient. Such a pharmaceutical composition may be administered to a patient, and the biological conjugate is permitted to contact the target to deliver the compound to the target in the patient. In these embodiments, useful pharmaceutically acceptable carriers include materials such as water, gelatin, lactose, starch, magnesium stearate, talc, plant oils, gums, alcohol, Vaseline, or the like. The pharmaceutical preparation of the invention should include an amount of the biological conjugate effective for the desired activity. The effective dosage will depend on the activity of the particular biological conjugate employed and is thus within the ordinary skill of the art to determine for any particular host mammal or other host organism.
In general, a pharmaceutically effective amount of the biological conjugate is combined in a conventional fashion with the pharamaceutically acceptable carrier to produce the pharmaceutical composition. The pharmaceutical composition of the invention is preferably administered internally, e.g., intravenously, in the form of conventional pharmaceutical preparations, for example in conventional enteral or parenteral pharmaceutically acceptable excipients containing organic and/or inorganic inert carriers as described above. The pharmaceutical preparations can be in conventional solid forms, for example, tablets, dragees, suppositories, capsules, or the like, or conventional liquid forms, such as suspensions, emulsions, or the like. If desired, they can be sterilized and/or contain conventional pharmaceutical adjuvants, such as preservatives, stabilizing agents, wetting agents, emulsifying agents, buffers, or salts used for the adjustment of osmotic pressure. The pharmaceutical preparations may also contain other therapeutically active materials.
EXAMPLES
The following Examples are intended to illustrate, but in no way limit the scope of the present invention. All parts and percentages are by weight and all temperatures are in degrees Celsius unless explicitly stated otherwise.
Example 1
Construction and use of Expression Vectors Containing C4-and a Linker Peptide
1. Bacterial Strains and Plasmids
E. coli strain DH5α-T1 is commercially available from Invitrogen (USA). E. coli strains B121(DE3)and NovaBlue are commercially available from Novagen (USA). The pET29a(+) bacterial expression vector was obtained from Novagen (USA). The pLen-121 plasmid containing the DNA sequence encoding the 121-amino acid residue isoform of human VEGF has been described in U.S. Pat. No. 5,219,739, herein incorporated by reference in its entirety, and was obtained from Dr. J. Abraham (Scios Nova, Inc., USA). The pPAP1-1.6 plasmid containing the DNA sequence encoding human annexin V was obtained from Dr. J. Tait (University of Washington School of Medicine, Seattle, Wash.). The pGEX-KG LF254 plasmid containing the DNA sequence encoding non-toxic N-terminal fragment of anthrax lethal factor (LFn) was obtained from Dr. S. Leppla (National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Md.).
2. DNA Manipulations
The restriction and modification enzymes employed herein are commercially available in the U.S. and were used according to the manufacturer's instructions. Preparation of competent cells, transformation, and bacterial medium were according to Sambrook et al. (J. Sambrook, E. F. Fritsch and T. Maniatis. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or according to the manufacturer's instructions. Purification of plasmids was done using Wizard Plus SV Minipreps or Maxipreps DNA Purification Systems (Promega, USA) according to the manufacturer's instructions. The purification of DNA fragments from agarose gels was done using the Geneclean Spin kit (Bio 101, USA) according to the manufacturer's instructions.
3. Construction of Vectors for Expression of Proteins Fused to C4-Tag.
The pET/Hu(G4S)3 vector for expression of recombinant proteins with an N-terminal Hu-tag fused to protein ORF via a (G4S)3 linker was constructed as described (Backer et al., 2004). The R4C amino acid substitution was introduced in Hu-tag of the pET/Hu(G4S)3 vector by site-directed mutagenesis using Gene-Tailor Site Directed mutagenesis kit (Invitrogen). Primers (SEQ ID NO:23 and SEQ ID NO:24) were used for introducing the R4C mutation. The resulted vector was confirmed by sequencing and designated pET/Hu-C4(G4S)3.








SEQ ID NO:23











5′-ATATACATATGAAAGAATCTTGCGCTAAAAAATTTC




 








SEQ ID NO:24











5′-AGATTCTTTCATATGTATATCTCCTTCTTAAAGTT







DNA encoding C4-G4S was amplified by PCR using pET/Hu-C4(G4S)3 vector DNA as a template, a sense primer SEQ ID NO:25 introducing XbaI site, and antisense primer SEQ ID NO:26 introducing NcoI site. The insert was cloned into the pET29a(+) vector cut with XbaI and NcoI. The resulting vector was confirmed by sequencing and designated pET/Hu-C4-G4S.








SEQ ID NO:25











5′-GGATAACAATTCCCCTCTAGAAATAATTTTGTTTAAC




 








SEQ ID NO:26











5′-ACTACCCATGGAACCGCCGCCACCAGAGTCCATG







4. Sub-Cloning of cDNA Fragments Encoding Proteins of Interest into the pET/Hu-C4(G4S)3 and pET/Hu-C4-G4S Vectors.
The pET/Hu-C4(G4S)3 and pET/Hu-C4-G4S vector DNAs were cut separately with NcoI and dephosphorylated. All DNA fragments to be cloned in the above vectors were amplified by PCR using primers introducing NcoI sites to the both ends of the amplified DNA fragments. DNA encoding the 121-aa isoform of human VEGF was amplified from the pLen-121 plasmid DNA using primers (SEQ ID NO:27 and SEQ ID NO:28). DNA encoding a 1-254-aa long N-terminal fragment of anthrax lethal factor (LFn) was amplified from the pGEX-KG LF254 plasmid DNA using primers (SEQ ID NO:29 and SEQ ID NO:30). DNA encoding human annexin V was amplified from the pPAP1-1.6 plasmid using primers (SEQ ID NO:31 and SEQ ID NO:32). Each amplified DNA fragment was cut with NcoI, purified, and cloned into NcoI sites of the vectors pET/Hu-C4(G4S)3 and pET/Hu-C4-G4S. Clones with correct orientations of cloned ORF were selected by PCR using a T7-promoter based sense primer, and confirmed by sequencing.








SEQ ID NO:27









5′-CACAAGCCATGGCACCCATGGCAGAAGGAGGA



 








SEQ ID NO:28









5′-ACTACCCATGGTCACCGCCTCGGCTTGTCAC



 








SEQ ID NO:29









5′-CTGCTCCATGGGAGCGGGCGGTCATGGTGATG



 








SEQ ID NO:30









5′-ACTACCCATGGCTATAGATTTATTTCTTGTTCGTTAAATTTATC



 








SEQ ID NO:31









5′-CACAAGCCATGGCACAGGTTCTCAGAG



 








SEQ ID NO:32









5′-ACTACCCATGGTTAGTCATCTTCTCCACAGAGC







5. Expression and Purification of Recombinant Fusion Proteins with C4-Tag.
All proteins were expressed in BL21(DE3) E. coli grown in LB medium (Q-Biogen, Carlsbad, Calif.). The expression was induced by 1 mM IPTG at the optical density of 0.5-0.7 unit at 600 nm. After induction, cultures were grown for 2.5-3 hrs at 37° C. with shaking at 300 rpm; then harvested by centrifugation and ruptured using EmulsiFlex-C5 (Avestin, Ottawa, Canada).
C4-VEGF was expressed in insoluble form and purified as follows: inclusion bodies were solubilized in 8 M urea, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 80 mM Na2SO3, 10 mM Na2S4O6, 10 mM DTT, incubated for 6-8 hrs at 4° C. with agitation; then supplemented with 2.5 mM Tris[2carboxyethyl]phosphine (Pierce), and incubated for 16-18 hrs at 4° C. Solubilized protein was refolded via a tree-step dialysis: first, 10-12 hrs at 4° C. in 10 volumes of 20 mM Tris-HCl pH 8.0, 2 M urea, 0.5 M arginine, 1 mM reduced glutathione, 0.4 mM oxidized glutathione; second, 24 hrs at 4° C. in 50 volumes of 20 mM Tris-HCl pH 8.0; and third, 24 hrs at 4° C. in 50 volumes of 20 mM NaOAc (pH 5.5). After dialysis, solution was clarified by centrifugation (15,000×g for 20 min), and C4-VEGF was purified by chromatography on Heparin HP Sepharose (1-ml pre-packed columns, Amersham). For conjugation, C4 thiol group in C4-VEGF was deprotected by mild DTT treatment using 1.2 molar excess DTT at 34° C. for 45 min.
To minimize potential involvement of cysteine residue present in position 116 in native VEGF 121, the inventors prepared a version of C4-VEGF truncated at position 112 using methods known in art. Truncated protein contains 110 amino acids (a 3-112aa fragment of VEGF121), and therefore it was named C4-VEGF110. The C4-VEGF110 was expressed and purified as described above and displayed activities similar to that of C4-VEGF in tissue culture assays.
To further optimize C4-tagged VEGF the inventors constructed a single chain VEGF fused to a single C4 peptide tag via a G4S linker peptide (C4-scVEGF). In general, engineering, expression, refolding, and purification of monomeric proteins is simplier than oligomeric, or even dimeric, such as native isoforms of VEGF. In addition, scVEGF was designed to avoid formation of inactive monomers of VEGF that are usually present in preparations of dimeric VEGF, to simplify any protein re-engineering of VEGF, to simplify expression, refolding and purification and to simplify conjugation of binding moieties to a single C4-tag. First, the pET/Hu-C4-G4S vector DNA was cut with BamHI and dephosphorylated. DNA encoding a 3-112-aa long fragment of human VEGF was amplified from the pLen-121 plasmid using a sense primer (SEQ ID NO:33) introducing BamHI site immediately upstream of VEGF codon 3, and an antisense primer (SEQ ID NO:34) introducing a stop codon immediately downstream of codon 112 of VEGF and BamHI site immediately after the stop codon. The amplified DNA fragment was purified, cut with BamHI, and cloned into BamHI site of the pET/Hu-C4-G4S vector. Correct orientation of cloned ORF was selected by PCR using a T7-promoter based sense primer. Plasmid DNA purified from the selected clones was confirmed by sequencing and designated pET/C4(G4S)VEGF110.








SEQ ID NO:33











5′-CACGGATCCGGTGGCGGCGGTAGTGGT




 








SEQ ID NO:34











5′-CACGGATCCTCATCTTGCTCTATCTTTCTTTGGTCTGC







The pET/C4(G4S)VEGF110 vector DNA was cut with NcoI and dephosphorylated. DNA encoding a 3-112-aa fragment of human VEGF was amplified from the pLen-121 plasmid by PCR using a sense primer (SEQ ID NO:35) introducing NcoI site immediately upstream of VEGF codon 3, and antisense primer (SEQ ID NO:36) introducing a cytosine immediately downstream of VEGF codon 112 (for compensation of ORF shift) and NcoI site immediately after the introduced cytosine. The amplified DNA fragment was purified, cut with NcoI, and cloned into NcoI site of the pET/C4(G4S)VEGF110 vector. Clones with two VEGF110 copies were selected by PCR using a T7-promoter based sense primer and a T7-terminator based antisense primer. DNA isolated from four random selected clones was sequenced, and a clone containing a VEGF110 tandem with a head-to-tail VEGF110 orientation was selected for propagation. The selected plasmid designated pET/C4(G4S)scVEGF was transformed into competent E. coli cells strain BL21 (DE3) for protein expression.








SEQ ID NO:35











5′-CACAAGCCATGGCACCCATGGCAGAAGGAGGA




 








SEQ ID NO:36











5′-ACTACCCATGGCTCTTGCTCTATCTTTCTTTGGTCTGC







The C4-scVEGF was expressed and purified as described above and displayed activities similar to that of C4-VEGF in tissue culture assays. C4-scVEGF was recovered with  ̃50% of C4 thiol group available for conjugation. For conjugation, C4 thiol group in C4-VEGF was deprotected by mild DTT treatment with 0.5 molar equivalent of DTT.
C4-LFn was expressed in a soluble cytoplasmic form and purified as follows: soluble part of bacterial lysate was dialyzed against 200 volumes of 20 mM Tris-HCl pH 8.0 for 20 hrs at 4° C., clarified by centrifugation (15,000 g for 20 min); and passed through Sepharose Q FF column. C4-LFn containing fractions were pooled together, dialyzed against 100 volumes of 20 mM NaOAc pH 6.5 for 20 hrs at 4° C., and purified on Heparin HP Sepharose followed by hydrophobic interaction chromatography on Butyl FF Sepharose (1-ml pre-packed columns, Amersham). The yield of C4-LFn was 8-10 mg/L, purity >98% by SDS-PAGE followed by SimplyBlue Safe Stain (Invitrogen).
C4-annexin was purified as described for Hu-annexin (Backer et al., 2004). Depending on the nature of a linker, and a protein, a cysteine residue in C4-tag is involved to a various degree in mixed disulfide with red-ox components of the refolding buffer. When necessary, cysteine residues can be deprotected with DTT under conditions that are optimized for every C4-protein.
6. Construction and Expression of Adapter Proteins.
6.1. Chimeric 1-29B/30-127H-RNase (BH-RNase) comprising of a 1-29 aa fragment of bovine RNase A and a 30-127 aa fragment of human RNase I has been constructed, expressed and purified as described (Gaynutdinov et al., 2003). The V118C mutation was introduced in the pET29/1-29B/30-127H-RNase plasmid DNA by site-directed mutagenesis using Gene-Tailor Site Directed mutagenesis kit (Invitrogen) and primers (SEQ ID NO:37 and SEQ ID NO:38). The VC118 substitution was confirmed by sequencing, the protein was designated BH-RNase(C118). BH-RNase(C118) was expressed and purified as described for wild type BH-RNase (Gaynutdinov et al, 2003). The presence of a reactive thiol group was tested by reaction with a thiol reagent N-(1-pyrene)-maleimide (Molecular Probes, Eugene, OR) followed by RP HPLC analysis as described (Backer et al., 2002). HuS(C118) adapter protein was obtained by limited digestion of BH-RNase(C118)with subtilisin (Sigma) as described for BH-RNase (Gaynutdinov et al., 2003).








SEQ ID NO:37











5′-AAGGCTCTCCGTACGTTCCGTGTCATTTCGACGCG




 








SEQ ID NO:38











5′-CGGAACGTACGGAGAGCCTTCGCATGCAAC







6.2. HuS(C88, C118)
The N88C mutation was introduced in the pET29/1-29B/30-127H-RNase(V118C) plasmid DNA by site-directed mutagenesis using primers (SEQ ID NO:39 and SEQ ID NO:40). The N88C substitution was confirmed by sequencing, and the protein was designated BH-RNase(C88, C118). BH-RNase(C88, C118) was expressed, purified and digested by subtilisin to yield HuS(C88, C118) adapter protein as described above for BH-RNase(C118).









5′-TCACTGACTGCCGTCTTACTTGCGGATCCCGTT
SEQ ID NO:39



 


8 5′-AGTAAGACGGCAGTCAGTGATATGCATAGAA
SEQ ID NO:40






6.3 HuR(A8, P11, C118)
The Q11P and F8A mutations were introduced consequently in the pET29/1-29B/30-127H-RNase(V118C) plasmid DNA by site-directed mutagenesis using primers SEQ ID NOS:41-44, respectively. Both substitutions were confirmed by sequencing, and the protein was designated BH-RNase(A8, P11,C118). BH-RNase(A8, P11, C118) was expressed and purified as described above for BH-RNase(C118).









5′-GCAGCCAAGTTTGAGCGGCCGCACATGGACTC
SEQ ID NO:41



 


5′-GCCGCTCAAACTTGGCTGCTGCAGTTTCCTT
SEQ ID NO:42


 


5′-GGAAACTGCAGCAGCCAAGGCTGAGCGGCCGC
SEQ ID NO:43


 


5′-CTTGGCTGCTGCAGTTTCCTTCATATGTATAT
SEQ ID NO:44






7. Construction and Expression of Proteins Used in the Assays.
7.1. SLT-VEGF.
The pET/VEGF121-SLT/L plasmid encoding SLT-VEGF fusion protein carrying N-terminal S-tag and His-tag was described previously (Backer & Backer, 2001). The SLT-VEGF DNA coding sequence was amplified by PCR from the pET/VEGF121-SLT/L plasmid DNA using primers (SEQ ID NO:45) introducing NdeI site and (SEQ ID NO:46) introducing Xho I site. Purified PCR product was cut with Nde I and Xho I restrictases, purified, and cloned into Nde I-Xho I sites of the pET29a(+) vector (Novagen). The resulting plasmid was confirmed by sequencing and designated pET29/SLT-VEGF. SLT-VEGF was expressed in BL21(DE3) as described above for C4-carrying proteins, and purified as follows: inclusion bodies were solubilized in 8 M urea, 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 80 mM Na2SO3, 10 mM Na2S4O6, 10 mM DTT and incubated for 6-8 hrs at 4° C. with agitation; then supplemented with 5 mM Tris[2carboxyethil]phosphine (Pierce) and incubated for 16-18 hrs at 4° C. Solubilized protein was refolded via a tree-step dialysis: first, for 8-10 hrs at 4° C. in 10 volumes of 20 mM Tris-HCl pH 8.0, 2 M urea, 0.5 M arginine, 1 mM reduced glutathione, 0.4 mM oxidized glutathione; second, 24 hrs at 4° C. in 100 volumes of 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% NP-40; and third, 24 hrs at 4° C. in 100 volumes of 20 mM Tris-HCl pH 8.0, 20 mM NaCl. After dialysis, solution was clarified by centrifugation (15,000×g for 20 min), and SLT-VEGF was purified by chromatography on HiTrap Q Sepharose (1-ml pre-packed columns, Amersham).








SEQ ID NO:45











5′- AAAAACATATGAAGGAATTTACCTTAGACTTCTCG




 








SEQ ID NO:46











5′- TACTCGAGTCACCGCCTCGGCTTGTCAC







7.2. Hu-VEGF.
Hu-VEGF 121 fusion protein carrying N-terminal wild type Hu-tag was constructed as described in Backer et al., 2003. Hu-VEGF121 was expressed and refolded from inclusion bodies as described above for C4-VEGF with the following modifications: refolding from inclusion bodies was done via a two-step dialysis: first, for 8-10 hrs at 4° C. in 10 volumes of 20 mM Tris-HCl pH 8.0, 2 M urea, 0.5 M arginine, 1 mM reduced glutathione, 0.4 mM oxidized glutathione; second, 24 hrs at 4° C. in 50 volumes of 20 mM Tris-HCI pH 8.0. Also, for final Hu-VEGF121 purification, HiTrap SP Sepharose FF followed by Heparin HP Sepharose chromatography (1-ml pre-packed columns, Amersham) was performed.
8. Functional Activities of Recombinant Fusion Proteins with C4-Tag.
A. VEGF activity. Functional activities of VEGF and VEGF-based conjugates were tested in two assays. First assay, stimulation of VEGFR-2 autophosphorylation was performed as follows: near-confluent 293/KDR cells after overnight starvation (DMEM/0.5% FBS) were shifted to serum-free DMEM with 0.5 mM sodium vanadate for 20 min at 37° C., then stimulated with VEGF for 10 min at 37° C., lysed and analyzed by Western blotting using anti-phosphotyrosine RC20:HRPO conjugate (BD Transduction Labs, USA). Second assay, protection of 293/KDR cells from cytotoxic effect of SLT-VEGF, was performed as follows. 293/KDR cells were plated on 96-well plates 20 hrs before the experiment, 1000 cells/well. Varying amounts of VEGF or VEGF-based conjugates were mixed with SLT-VEGF in complete culture medium, and added to cells in triplicate wells to a final SLT-VEGF concentration of 1 nM. Viable cells were quantitated 96 hrs later by CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega, USA).
B. Annexin V activity. Functional activities of C4-annexin and HuS-C4-annexin were tested by their ability to compete with FITC-annexin (Sigma) for binding to phosphatidylserine-displaying erythrocytes of stabilized human blood (4 C Plus Cell Control, Beckman Coulter, USA) as described (Tait et al., 1995). Briefly, ten million erythrocytes were incubated with varying amounts of annexin in the presence of 5 nM FITC-annexin V (Sigma, St. Louis, Md.) in binding buffer (10 mM HEPES, pH 7.4, 136 mM NaCl, 2.5 mM CaCl2) for 15 min at RT. Cells were spun down, resuspended in a binding buffer supplemented with 5 mM EDTA, and spun down again. FITC-annexin fluorescence in the supernatants was measured at λex 485 nm/λem 520 nm.
C. LFn activity. C4-LFn and Hus-C4-LFn were tested for their ability to protect RAW cells from cytotoxic effects of full-length LF in the presence of PA. RAW264.7 cells were plated on 96-well plates 15×103 cells/well 20 hrs before the experiment. Varying amounts of LFn, C4-LFn, or Hus-C4-LFn were mixed with LF and PA (List Biological, USA) in DMEM complete culture medium, and added to cells in triplicate wells to final concentrations of 2 nM PA and 0.2 nM LF. After 2.5 hrs of incubation at 37° C. in 5% CO2, viable cell numbers were determined by CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega).
9. Cell Lines.
HEK293 human transformed embryonic kidney cells (CRL-1573) and RAW 264.7 mouse monocytes (TIB-71) were from American Type Culture Collection (ATCC, Rockville, Md.). 293/KDR cells expressing 2.5×106 VEGFR-2/cell have been developed in SibTech, Inc. (Newington, Conn.; Backer and Backer, 2001a). All cells were maintained in DMEM (Life Technologies, USA) supplemented with 10% fetal calf serum (Gemini, USA), 2 mM glutamine (Life Technologies, USA), and penicillin-streptomycin (Life Technologies, USA) at 37° C., 5% CO2.
Example 2
Site-Specific Conjugation of C4-Tagged Proteins to Adapter Protein HuS(C118)
The protocol included site-specific conjugation of C4-tagged proteins to adapter protein HuS(C118) and testing the functional activities of conjugates in tissue culture.
HuS(C118) prepared as described above, and C4-tagged proteins, such as C4-VEGF, or C4-annexin, or C4-LFn, were mixed in a buffer containing 20 mM Tris HCI pH 8.0, incubated at 4° C. for 16 hrs, and then analyzed by SDS-PAGE under reducing and non-reducing conditions. SDS-PAGE analysis under non-reducing conditions revealed new protein products formed in the mixtures during incubation (FIG. 2C, no DTT lanes). The molecular weights of the proteins in these bands corresponded to the molecular weight of conjugates carrying one or two HuS molecules per parental protein, respectively. Under reducing conditions these bands disappeared, and only bands corresponding to parental proteins and 12-kDa HuS(C118) were detectable (FIG. 2C, plus DTT lanes). Conjugates were purified first on Hu-peptide column to remove free HUS(C118) and, when necessary, by ion-exchange chromatography to remove free parental proteins.
The functional activities of conjugates were tested in tissue culture assays described above. Activity of HuS-C4-VEGF conjugate was similar to that of C4-VEGF in the induction of VEGFR-2 tyrosine autophosphorylation in 293/KDR cells (FIG. 3, Panel A) HuS-C4-annexin conjugate was active in competition with FITC-annexin for binding to phosphatidylserine-displaying erythrocytes of stabilized human blood with IC50 of 11+3 nM for conjugate vs. IC50 of 9+4 nM for recombinant annexin V as reported by Tait et al., (1995) (FIG. 3, Panel B). HuS-C4-LFn was as active as C4-LFn in protection of RAW 264.7 cells from LF in the presence of PA (FIG. 3, Panel C). Together, these data indicate that conjugation of adapter protein to different C4-tagged protein does not destroy their activity.
Example 3
Site-Specific Conjugation of C4-VEGF and C4-scVEGF to Radionuclide Chelator, 5-Maleimido-2-Hydraziniumpyridine Hydrochloride
The protocol included site-specific conjugation of C4-VEGF and C4-scVEGF to chemically active radionuclide chelator 5-maleimido-2-hydraziniumpyridine hydrochloride (Solulink, San Diego, Calif.), and testing the conjugate in tissue culture.
C4-VEGF, prepared as described above, was mixed with dimethylformamide dissolved 5-maleimido-2-hydraziniumpyridine hydrochloride hereinafter designated as HYNIC at the molar ratio HYNIC/protein 3:1 in a buffer containing 20 mM Tris HCl pH 8.0 and incubated for one hour at room temperature. The product, designated HYNIC-C4-VEGF, was purified on PD-10 column (Amersham, USA) equilibrated with 0.114 M Tricine, pH 6.9. Analytical RP HPLC (FIG. 4, Panel A) was used to determine the concentration of protein by detection of optical density at 216 nm and the concentration of HYNIC by detection of optical density at 310 nm. Under selected conditions the ratio of HYNIC to C4-VEGF in purified HYNIC-C4-VEGF was  ̃1.
C4-scVEGF, prepared as described above, was conjugated to HYNIC and purified as described above and the ratio of HYNIC to C4-scVEGF in purified product designated HYNIC-C4-VEGF, was  ̃1.
The functional activity of VEGF moiety in HYNIC-C4-VEGF and HYNIC-C4-scVEGF was tested in two tissue culture assays described above, induction of VEGFR-2 tyrosine autophosphorylation in 293/KDR cells(FIG. 4, Panel B) and protection of 293/KDR cells from cytotoxic activity of SLT-VEGF (FIG. 4, Panel C). In both assays activities of HYNIC-C4-VEGF and HYNIC-C4-scVEGF were comparable to that of unmodified C4-VEGF, indicating that conjugation of C4-VEGF or C4-scVEGF to HYNIC does not destroy activity of the protein.
Example 4
Site-Specific Conjugation of C4-VEGF to Polyethyleneglycol
The protocol included site-specific conjugation of C4-VEGF110 to polyethyleneglycol functionalized with chemically active maleimide group (Nektar Therapeutics) and testing the conjugate in tissue culture.
C4-VEGF110, prepared as described above, was mixed with either 20 kDa or 40 kDa polyethyleneglycol maleimide (PEG) at PEG to protein ratio of 3:1 and incubated for one hour at room temperature in a buffer containing 20 mM Tris HCl pH 8.0. The products, designated PEG20-C4-VEGF and PEG40-C4-VEGF, were purified from unreacted C4-VEGF and PEG by HPLC gel-filtration on a column equilibrated with 20 mM Tris HCI pH 8.0. Analytical RP HPLC was used to determine the concentration of protein by detection of optical density at 216 nm. Western blot analysis of reduced PEGylated VEGF products with antibody against VEGF revealed a band corresponding to apparent molecular mass of 55 kDa and a band of approximately equal intensity corresponding to unmodified VEGF monomer (FIG. 5, Panel A), indicating that in the majority of the VEGF dimers only one C4-tag was conjugated to PEG.
The functional activity of PEG-C4-VEGF was tested in tissue culture assay of induction of VEGFR-2 tyrosine autophosphorylation in 293/KDR cells (FIG. 5, Panel B for PEG40-C4-VEGF) In this assay activities of PEG-C4-VEGF were comparable to that of unmodified C4-VEGF, indicating that site-specific conjugation of C4-VEGF to polyethyleneglycol does not destroy activity of the protein.
Example 5
Site-Specific Conjugation of Cyanine Dye Cy5.5 To HuS-C4-VEGF Conjugate
The protocol included preparation of HuS(C88, C118), its conjugation to C4-VEGF, purification of the resulting Hus-C4-VEGF conjugate, and conjugation of said conjugate to a cyanine dye Cy5.5 yielding conjugate named Cy5.5-Hus-C4-VEGF (FIG. 6, Panel A). For comparative purposes, C4-VEGF was either randomly modified with NHS-Cy5.5 on amino groups to the ratio of 1:1, or modified with maleimide-Cy5.5 on C4 residue in the C4-tag. The functional activities of VEGF moiety in all Cy5.5-containing conjugates were tested in two tissue culture assays described above, induction of VEGFR-2 tyrosine autophosphorylation (FIG. 6, Panel B) and protection of 293/KDR cells from cytotoxic activity of SLT-VEGF (FIG. 6, Panel C). In both assays only activities of Cy5.5-Hus-C4-VEGF conjugate were comparable to that of unmodified C4-VEGF, underlying efficacy of using an adapter protein capable of binding to C4-tag for derivatization of proteins.
The utility of Cy5.5-Hus-C4-VEGF for imaging was tested in Balb/c female mice bearing subcutaneous 4T1 mouse mammary adenocarcinoma tumors. Images obtained with KODAK Image Station 2000 MM equipped with a band-pass filter at 630 nm and a long-pass filter at 700 nm indicated that Cy5.5-HuS-C4-VEGF conjugate preferentially localized to the periphery of primary tumor (FIG. 6, Panel D). This preferential localization was inhibited in mice pretreated with SLT-VEGF protein that destroys VEGFR-2 positive cells in tumors (Backer et al., submitted), indicating that accumulation of Cy5.5-HuS-C4-VEGF conjugate in tumor is VEGF-receptor mediated and can be used for specific imaging of tumor vasculature.
Example 6
The Use of C4-Tag and an Adapter Protein Covalently Bound to this Tag for Construction of VEGF-Driven Conjugates Containing Drug-Loaded Liposomes for Targeted Drug Delivery
The protocol included preparation of HuS(C118), its conjugation to PEG-lipid-maleimide, insertion of lipidated HuS(C118) into doxorubicin-loaded liposomes, that leads to a standardized construct named Lip/HuS(C118), and conjugation of this construct to C4-VEGF, that yielded Lip/Hus-C4-VEGF (FIG. 7, Panel A). The functional activity of VEGF moiety in Lip/Hus-C4-VEGF conjugate were tested in a tissue culture assay described above, induction of VEGFR-2 tyrosine phosphorylation in 293/KDR cells (FIG. 7, Panel B). In this assay VEGF activity of Lip/Hus-C4-VEGF liposomes was comparable to that of unmodified C4-VEGF, underlying efficacy of using an adapter protein capable of binding to C4-tag for derivatization of proteins.
A 15-min exposure of 293/KDR cells to Lip/HuS-C4-VEGF resulted in a dose-dependent inhibition of cell growth (FIG. 7, Panel C), while equivalent amounts of untargeted doxorubicin-loaded liposomes (commercially available under the trade name “DOXIL”) derivatized with HuS(C118) alone were not toxic for these cells indicating a VEGF receptor-mediated mechanism of cytotoxicity of Lip/Hus-C4-VEGF targeted liposomes. This mechanism was further confirmed by ability of VEGF to inhibit of cytotoxicity of Lip/Hus-C4-VEGF in a dose-dependent manner (FIG. 7D).
Example 7
The Use of C4-Tag for Construction of Scvegf-Driven Conjugates Containing Drug-Loaded Liposomes for Targeted Drug Delivery.
The protocol included preparation of C4-scVEGF its conjugation to PEG-lipid-maleimide, and insertion of lipidated C4-scVEGF into doxorubicin-loaded liposomes (“DOXIL”) (FIG. 8, Panel A). The functional activity of VEGF moiety in Lip/C4-scVEGF conjugate were tested in a tissue culture assay described above, induction of VEGFR-2 tyrosine phosphorylation in 293/KDR cells (FIG. 8, Panel B). In this assay VEGF activity of Lip/C4-scVEGF liposomes was comparable to that of unmodified C4-VEGF, underlying efficacy of using C4-scVEGF.
A 15-min exposure of 293/KDR cells to Lip/C4-scVEGF resulted in a dose-dependent inhibition of cell growth (FIG. 8, Panel C), while equivalent amounts of untargeted doxorubicin-loaded liposomes (“DOXIL”) were not toxic for these cells indicating a VEGF receptor-mediated mechanism of cytotoxicity of Lip/C4-scVEGF targeted liposomes. This mechanism was further confirmed by ability of VEGF to inhibit of cytotoxicity of Lip/Hus-C4-VEGF in a dose-dependent manner (FIG. 8D).
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## Claims

1. A biological conjugate, comprising:
(a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein selected from the group consisting of human vascular endothelial growth factor (VEGF) comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 8, human annexin V comprising the sequence of SEQ ID NO: 10, a catalytically inactive fragment of anthrax lethal factor comprising the sequence of SEQ ID NO: 12, and a single chain vascular endothelial growth factor (scVEGF) comprising the protein sequence of SEQ ID NO: 4 or SEQ ID NO: 14; and
(b) a binding moiety selected from the group consisting of drugs, radionuclide chelators, polyethylene glycol, dyes, lipids, liposomes, a surface of a nano- or microparticle, a surface of a dendrimer, a surface of a tissue scaffold, a surface of a biomedical device, and a surface of a biosensor;
said biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and said binding moiety.

2. The biological conjugate of claim 1, wherein said single chain vascular endothelial growth factor (scVEGF) comprises the sequence of SEQ ID NO:14.

3. A biological conjugate, comprising:
(a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein selected from the group consisting of human vascular endothelial growth factor (VEGF) comprising the sequence of SEQ ID NO: 6 or SEQ ID NO: 8, human annexin V comprising the sequence of SEQ ID NO: 10, a catalytically inactive fragment of anthrax lethal factor comprising the sequence of SEQ ID NO: 12, and a single chain vascular endothelial growth factor (scVEGF) comprising the protein sequence of SEQ ID NO: 4 or SEQ ID NO: 14; and
(b) a binding moiety comprising a complimentary adapter protein covalently bound to said targeting moiety, and wherein said adapter protein is a fragment of mutant human Rnase I comprising Cys118;
said biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and said binding moiety.

4. The biological conjugate of claim 3, wherein said adapter protein has a polypeptide sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:22.

5. A pharmaceutical composition comprising:
(a) a pharmaceutically acceptable carrier; and'
(b) the biological conjugate of claim 1.