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.

Description of the Invention

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.

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.

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 .alpha.-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:

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 VEGF.sub.110, 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 VEGF.sub.121 or VEGF.sub.110, 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 (VEGF.sub.121) 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 VEGF.sub.121 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 Gly.sub.4Ser or (Gly.sub.4Ser).sub.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 (see Original Patent), 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 (Cys.sub.118). 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 (see Original Patent) 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.
 

Claim 1 of 5 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.
 

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