Crosslinking of proteins and other entities via conjugates of α-haloacetophenones, benzyl halides, quinones, and their derivatives

Abstract

The present invention relates to the formation of conjugates (e.g., protein-protein dimers) using a-halo-acetophenones, benzylic halides, quinones, and related compounds as a conjugating system. The invention also features compositions that include the conjugates described herein, as well as uses of these conjugates in methods of medical treatment.

Claims

1. A conjugate having the following structure, ##STR00095## wherein A is optionally substituted aryl, optionally substituted heteroaryl, or a substructure according to the formula —Ar.sup.Z1—Z—Ar.sup.Z2—, wherein each of Ar.sup.Z1 and Ar.sup.Z2 is, independently, optionally substituted aryl or optionally substituted heteroaryl, and Z is a covalent bond, O, S, NR.sup.Z1, wherein R.sup.Z1 is H or optionally substituted C1-6 alkyl, an optionally substituted C1-20 alkylene, a polyethylene glycol of the structure (CH.sub.2CH.sub.2O)(CH.sub.2CH.sub.2O).sub.n(CH.sub.2CH.sub.2), wherein n is an integer between 0-1000, or a linking group having the structure —X.sup.Z1-(Q.sup.Z1).sub.n3-R.sup.Z2-(Q.sup.Z1).sub.n4 X.sup.Z1, wherein each of n3 and n4 is, independently, 0 or 1, each X.sup.Z1 is, independently, a covalent bond, O, S, or NR.sup.Z1, each Q.sup.Z1 is, independently, C(═O), S(═O), or S(═O).sub.2, and R.sup.Z2 is optionally substituted C1-20 alkylene or polyethyleneoxide (CH.sub.2CH.sub.2O)(CH.sub.2CH.sub.2O).sub.n(CH.sub.2CH.sub.2), wherein n is an integer between 0-1000; each of L.sup.1 and L.sup.2 is, independently, a covalent bond or optionally substituted C1-C6 alkylene; Q.sup.1 is a covalent bond, C(═O), S(═O).sub.2, or C1 alkylene; Q.sup.2 is C(═O) or S(═O).sub.2; each of n1 and n2 is, independently, 0 or 1; X.sup.1 is S; X.sup.2 is O, S, or NR.sup.X, wherein R.sup.Xis H or optionally substituted C1-6 alkyl; each R.sup.n is, independently, H or optionally substituted C1-6 alkyl; R is a globular protein; and R″ is a protein, a biologically active agent, or a biologically compatible agent; wherein each said protein independently comprises at least three amino acids linked by peptide bonds; and wherein optional substituents, when present, are independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, heteroaryl, —F, —Cl, —Br, —I, —N.sub.3, —NO.sub.2, —CN, —OC(═O)R.sup.B, —C(═O)R.sup.B, —OR.sup.B, —NR.sup.BC(═O)R.sup.C, —C(═O)NR.sup.AR.sup.B, —NR.sup.AR.sup.B, —CO.sub.2H, —CO.sub.2R.sup.B, —OC(═O)NR.sup.BR.sup.C, —NRC(═O)OR.sup.B, —OH, —NC, —S(═O).sub.2OR.sup.A, —S(═O).sub.2NR.sup.AR.sup.B, —NR.sup.AS(═O).sub.2R.sup.B, and —S(═O).sub.2R.sup.A, wherein each of R.sup.A, R.sup.B, and R.sup.C is independently H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, or heteroaryl.

2. The conjugate of claim 1, wherein L.sup.1 is a covalent bond; L.sup.2 is a covalent bond; n1 is 1; n2 is 1; and Q.sup.1 is C(═O), S(═O).sub.2, or CH.sub.2.

3. The conjugate of claim 1, wherein R″ is a protein.

4. The conjugate of claim 1, wherein one or both of R and R″ is a protein that is an annexin protein, an antibody, a cytokine, a wild-type protein that includes a free cysteine residue, or a protein modified to include a free cysteine residue, or wherein R″ is a biologically active or biologically compatible agent that is a polymer, nucleic acid, carbohydrate, small molecule therapeutic agent, imaging agent, or diagnostic agent.

5. The conjugate of claim 1, wherein A is optionally substituted phenyl, optionally substituted naphthyl, or optionally substituted biphenyl.

6. The conjugate of claim 1, wherein said conjugate has a structure according to one of the following formulas, ##STR00096##

7. A method of preparing a conjugate having a structure according to claim 1, comprising (a) contacting a compound according to the following formula, ##STR00097##  wherein A is optionally substituted aryl, optionally substituted heteroaryl, or a substructure according to the formula —Ar.sup.Z1—Z—Ar.sup.Z2—, wherein each of Ar.sup.Z1 and Ar.sup.Z2 is, independently, optionally substituted aryl or optionally substituted heteroaryl, and Z is a covalent bond, O, S, NR.sup.Z1, wherein R.sup.Z1 is H or optionally substituted C1-6 alkyl, an optionally substituted C1-20 alkylene, a polyethylene glycol of the structure (CH.sub.2CH.sub.2O)(CH.sub.2CH.sub.2O).sub.n(CH.sub.2CH.sub.2), wherein n is an integer between 0-1000, or a linking group having the structure —X.sup.Z1-(Q.sup.Z1).sub.n3-R.sup.Z2-(Q.sup.Z1).sub.n4 X.sup.Z1, wherein each of n3 and n4 is, independently, 0 or 1, each X.sup.Z1 is, independently, a covalent bond, O, S, or NR.sup.Z1, each Q.sup.Z1 is, independently, C(═O), S(═O), or S(═O).sub.2, and R.sup.Z2 is optionally substituted C1-20 alkylene or (CH.sub.2CH.sub.2O)(CH.sub.2CH.sub.2O).sub.n(CH.sub.2CH.sub.2), wherein n is an integer between 0-1000; each of L.sup.1 and L.sup.2 is, independently, a covalent bond or optionally substituted C1-C6 alkylene; C(═O), S(═O).sub.2, or C1 alkylene; Q.sup.2 is C(═O) or S(═0).sub.2; each of n1 and n2 is, independently, 0 or 1; each R.sup.n is, independently, H or optionally substituted C1-6 alkyl; and each of LG.sup.1 and LG.sup.2 is, independently, a leaving group, with a nucleophilic compound having the structure RX.sup.1H, wherein R is a globular protein, and X.sup.1 is S; and (b) contacting the product of step (a) with a nucleophilic compound having the structure R″X.sup.2H, wherein R″ is a protein or a biologically active or biologically compatible agent, and X.sup.2 is O, S, or NR.sup.X, wherein RX is H or optionally substituted C1-6 alkyl; wherein each said protein independently comprises at least three amino acids linked by peptide bonds; and wherein optional substituents, when present, are independently selected from the group consisting of C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, heteroaryl, —F, —Cl, —Br, —I, —N.sub.3, —NO.sub.2, —CN, —OC(═O)R.sup.B, —C(═O)R.sup.B, —OR.sup.B, —NR.sup.BC(═O)R.sup.C, —C(═O)NR.sup.AR.sup.B, —NR.sup.AR.sup.B, —CO.sub.2H, —CO.sub.2R.sup.B, —OC(═O)NR.sup.BR.sup.C, —NRC(═O)OR.sup.B, —OH, —NC, —S(═O).sub.2OR.sup.A, —S(═O).sub.2NR.sup.AR.sup.B, —NR.sup.AS(═O).sub.2R.sup.B, and —S(═O).sub.2R.sup.A, wherein each of R.sup.A, R.sup.B, and R.sup.C is independently H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl, or heteroaryl.

8. A pharmaceutical composition comprising the conjugate of claim 1 and a pharmaceutically acceptable excipient.

9. A method of delivering a therapeutic agent to a cell undergoing necrosis or apoptosis, said method comprising contacting said cell or tissue with an agent that is the conjugate of claim 1.

10. The method of claim 9, wherein one of R and R″ is an annexin protein.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The methods of conjugate formation described herein employ the specificity of α-haloacetophenone moieties, benzylic halides, quinones, and related electrophilic functional groups for addition reactions and displacements by nucleophiles (e.g., free thiol groups). The methods also employ the potential for useful secondary reactions in certain systems that augment the stability of the initial product. Conjugates prepared according to these methods can include various groups. For example, macromolecules (e.g., polymers such as polyethylene glycols, nucleic acids, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, or polysaccharides) such as polysialic acid, proteins conjugated as homo or hetero-dimers, or proteins conjugated to various small molecule therapeutic, imaging, diagnostic, or optical agents) can be used or prepared in the methods described herein. The present invention further relates to methods of making and using the conjugates themselves or as components in microarrays, the production of fine chemicals and kits, radio-labeling, molecular and optical imaging applications, and the diagnosis and treatment of disorders. In some embodiments, protein conjugates (e.g., those that include one or more annexin proteins) can also be used in these applications.

(2) Conjugates

(3) The methods described herein can be used to prepare conjugates that include a bifunctional linker derived from α-halocarbonyl, alkaryl halide, and quinone reagents, and related moieties. Exemplary conjugates can include a protein (e.g., a protein that is an antibody or a protein modified to include a free cysteine residue) or a biologically active or biologically compatible agent (e.g., polymer, a nucleic acid, carbohydrate, small molecule therapeutic agent, imaging agent, or diagnostic agent).

(4) The methods described herein employ nucleophilic groups (e.g., amino, hydroxyl, or thio functional groups) on the, e.g., proteins, biologically active agents, or biologically compatible agents that can react with electrophilic groups such as α-halo carbonyls, alkaryl halides, quinones, or electrophiles derived from carboxylic and sulfonyl functional groups. Thiol nucleophiles (e.g., cysteine residues) are particularly useful in these methods. If a suitable functional group is not present or not available for reaction with an electrophile, the compound to be conjugated can be modified according to methods known in the art that permit the introduction of an appropriate nucleophilic group (for example, a protein can be modified using protein synthesis methods known in the art to introduce, e.g., a cysteine residue) while retaining any beneficial properties (e.g., therapeutic activity) of the compound.

(5) Protein Conjugates

(6) Chemical crosslinkers and bioconjugation reagents are valuable tools that have been used for a variety of purposes, especially to link biological macromolecules and diverse polymers to both small and large molecular entities whose properties may be enhanced by such combinations. Prominent applications include covalent protein crosslinking techniques to conjugate antibodies, polyethylene glycol polymers, or imaging agents, immobilize ligands, attach haptens to carrier proteins, and stabilize folded protein structures and protein interaction complexes. The present invention applies to all such applications in which thiol groups can be exploited in selective reactions with α-halo-acetophenones and α-haloalkylbenzene derivatives.

(7) Protein-Protein Dimers

(8) Protein dimerization is a natural phenomenon that has important functional consequences in a variety of contexts. Protein-protein interactions are important for regulating functions and for transport across membranes. A number of proteins self-associate to form dimers within protein networks and cascades. (Marianayagam et al., TIBS, 27, 618-625, 2004). Receptor dimerization has been established as a general mechanism for the initiation of signal transduction, and many cell-surface receptors are believed to be activated by such a process, (Rodriguez-Frade et al., Trends in Immunology, 22:612-617, 2001; Wang et al., Annu. Rev. Immunol. 27:29-60, 2009). These interactions, however, are most often noncovalent and transient. Covalent dimers have been produced synthetically either by disulfide formation from the oxidation of protein thiols or by standard crosslinking techniques (Bioconjugate Techniques, Academic Press, New York, 2nd edition, 2008, (Au: G. T. Hermanson) using organic chemical coupling reactions. However, the crosslinking reactions that have been employed are usually not specific and, lacking a specific biochemical driving force that has evolved for functional reasons, are often quite slow in driving protein dimerization to completion (Ashworth et al., J Cell Sci. 112:3549-58, 1999). Unnatural covalent dimers that have been produced synthetically by disulfide formation often possess short lifetimes in vivo.

(9) Two factors that impose barriers to dimerization are the limiting solution concentrations of proteins required for homogeneous reaction media, and steric compression resulting from forcing large entities into close proximity. These conditions are often difficult to overcome in actual practice. One strategy is to employ the chemistry of thiol groups to form protein dimers. Disulfide motifs have been used in many instances for coupling biological entities, since the thiol group lends itself to selective redox modifications that do not interfere with other protein functional groups. In principle, with knowledge of structure, cysteines can be installed in each monomeric component and oxidation reactions can create disulfide-linked dimers; specificity, however, can be difficult to achieve when multiple cysteines are present. Cross-linking by bis-maleimide reagents represents another tack. Recently, Rotkoski et al. (Bioconjug Chem. 21:1691-702, 2010) have used bis- and tris-maleimides in conjunction with variants of ribonuclease A to form dimers and trimers of these proteins. The generality of this method is presently unclear and, further, each condensation to form thio-succinimides creates a new enantiomeric center that may produce complex mixtures that are difficult to resolve into separate components. These reagents have been most successful when the target entities are in close proximity, as in the case of protein subunits or in membranes. Additionally, maleimide-thiol adducts are unstable over time. This property limits their utility in therapeutics as both duration of action and storage properties are compromised.

(10) Given the limitations of forming stable disulfide dimers in vivo, and the apparent difficulty of preparing homogeneous dimers site specifically using other coupling methods, efficient methods for site-specific protein dimer formation would be useful. Although fusion proteins prepared by recombinant techniques are known in the art and are useful as therapeutics, chemically produced fusions are not limited to fusions at protein termini and can adapt to a wide variety of chemical structures and crosslinks for the preparation of target conjugates. Thus, increasing the range of novel chemical structures by the introduction of chemical modification techniques expands the fusion protein playing field, currently dominated by molecular biology approaches that are limited to protein termini and peptide crosslinks (Schmidt, Curr Opin. in Drug Discovery & Development, 12:284-295, 2009).

(11) To effect site-specific protein dimer formation, two steps are generally necessary to crosslink the component proteins. It is advantageous if the first step can be carried out by using a large excess of the small molecule conjugating reagent so that the rate of formation of the intermediate mono-conjugate can be selectively formed at a practical rate, as exemplified by α-halo-acetophenone methods described herein. The second dimer forming step may be more challenging as the protein concentrations may be difficult to manipulate due to the need to balance solubility with concentration and rate considerations. Therefore, intrinsically rapid coupling reactions are clearly desirable for conjugating proteins in relatively dilute solutions, and these are often at odds with the demands of site-selectivity.

(12) α-Halocarbonyl and Related Conjugates

(13) α-Halocarbonyl reagents, activated halide reagents (e.g., alkaryl halides such as benzyl halide), and related species are known alkylating agents that are capable of labeling amino- and thio-nucleophiles. For example, phenacyl bromides have been used to modify protein nucleophiles. We have discovered that a number of α-halocarbonyl and alkaryl halide species are capable of site-selectively labeling protein cysteine thiols. In one embodiment, we have exploited this preference to selectively prepare protein conjugates by using bifunctional groups that include an additional functional group QO.sub.2H that can be independently modified, chemospecifically by compounds of the structure RYH, where R represents, e.g., a protein, a biologically active agent, or a biologically compatible agent and YH represents a nucleophilic functional group such as hydroxy, thio, or amino (Scheme 1).

(14) ##STR00057##

(15) In Scheme 1 and other schemes described herein, substructure A can be selected from a variety of groups, including aryl (e.g., phenyl) and heteroaryl groups. In other embodiments, substructure A is, e.g., optionally substituted naphthyl, by analogy to the exemplary phenacyl chemistry described herein. In still other embodiments, substructure A may represent optionally substituted furan, thiophene, or other non-nucleophilic heterocycles containing two or more heteroatoms capable of bearing protein and biologically active or biologically compatible agents. Additionally, substructure A can include an aryl or heteroaryl group that is linked to another aryl or heteroaryl group via a covalent bond or a molecular linker (e.g., a C1-20 alkyl group, a bis-thioether group, or a polyethylene glycol linker). Exemplary, non-limiting substructures A are shown below in Scheme 2.

(16) ##STR00058## ##STR00059##
The linking group Z can be, for example, a covalent bond, an optionally substituted C1-20 alkylene group, a polyethyleneglycol group, or a C1-C20 dithioether group. The groups of substructure A, including those shown in Scheme 2, can include additional substituent groups.

(17) For example, in various embodiments, the thiol moieties of free cysteines of proteins (e.g., compounds represented by the formula “PSH”) can undergo site-specific alkylation reactions with halo acetyl groups linked to e.g., aromatic or heteroaromatic rings, by displacement of the halide, to form the corresponding protein thioethers as shown in Scheme 3. In some embodiments, the thioether can be part of a phenacyl system (substructure A=phenyl) in which the substituents can independently carry protein and biologically active or biologically compatible agents as part of sulfonamides, carboxamides, esters, and sulfones, etc. These intermediates can be prepared via reactions of appropriately constructed protein and biologically active or biologically compatible agents by reactions known in the art to activate carboxylic acids, sulfonic acids and the like.

(18) ##STR00060##
Similar procedures can be used for bifunctional groups having an alkaryl halide group (i.e., compounds of Scheme 1, where m=0).

(19) In other embodiments, the bifunctional linker group includes only α-halocarbonyl or benzylic halide functional groups as exemplified by Scheme 4, where “PSH” represents a protein, biologically active agent, or biologically compatible agent that includes a nucleophilic thiol group. These reagents can be used to prepare symmetrical conjugates (that is, conjugates where both P groups are the same compound) or asymmetrical conjugates (conjugates where each P group is a different compound).

(20) ##STR00061##

(21) In various embodiments, a nucleophilic group, e.g. a cysteine thiol group, in a protein can undergo alkylation reactions with α-halo-ketones or alkaryl halides to form, e.g., the corresponding thioethers. Reaction of haloketones and alkaryl halides can occur with oxygen, nitrogen, and sulfur nucleophiles (e.g., Erian et al., Molecules 8:793-865, 2003). Molecules containing α-bromoacetyl moieties are known to react preferentially with cysteine and methionine residues of proteins. The corresponding chlorides tend to be less reactive than the bromides, and the iodides are more reactive. We have in a broad survey established that bromoacetyl and chloroacetyl moieties linked to aromatic systems are sufficiently activated for alkylation of protein thiols. They, along with the corresponding iodides, are attractive substrates since such α-haloacetophenones bearing amide or sulfonamide ring substituents can be obtained commercially and/or prepared synthetically by those skilled in the art. Commercially useful protein and biologically active or biologically compatible agents (e.g., polyethylene glycol polymers or small molecule therapeutics) can be selectively linked through activated carboxy or sulfonoxy substituents without disturbing the bromo acetyl group or other halocarbonyls as described herein.

(22) In schemes described herein, PSH can be a protein containing at least one free thiol such as natural proteins such as annexin proteins, α-1-antiprotease, human sonic hedgehog N-terminal protein, oncostatin M, primary ribosomal protein S4, and other targetable free cysteines (e.g., annexins I, III, IV, V, VI and VIII, V-128). The present invention can also be applied to antibodies, minibodies, diabodies, affibodies and the like, and mutant proteins in which amino acid residues have been mutated to cysteines.

(23) In a general method, the protein is dissolved in a suitable solvent or buffer. A desired amount of the protein solution is incubated with the α-halo-carbonyl species for about 1-10 hours at a pH of about 6-10 and at a temperature of about 10-60° C. After the incubation is complete, the solution is subjected to centrifuge filtration. Removal of the excess reagents followed by optional purification provides the corresponding 2-thioalkyl-acetophenone systems or its analogs.

(24) In certain embodiments, the chemoselective conversion of the carbonyl species to oximes using aminoxy substrates may be carried out. For example, as shown in Scheme 5, condensation of bifunctional reagents with aminoxy substrates containing an R″ group that is, e.g., a protein, a biologically active agent, or a biologically compatible agent, results in the conjugation of an additional protein, biologically active agent, or biologically compatible agent to the protein target. A corresponding mono-oxime conjugate containing an aminoxy terminal side chain (from reaction of a bis-aminoxy substrates and the like) in lieu of a protein and biologically active or biologically compatible agents (not shown) would permit further modifications of the conjugated protein via a three step process. These reagents can be prepared according to procedures analogous to those described in U.S. patent application Ser. No. 13/030,772, International Patent Application No. PCT/US2011/025413, and PCT Publication No. WO2010/040147, each of which is hereby incorporated by reference.

(25) ##STR00062##
Alternatively, the alpha thiol carbonyl conjugate, the primary protein conjugate, may convert to a stable secondary product in which the alpha carbonyl is further reacted with a proximal protein nucleophile, that confers additional stability to the system as represented in Scheme 1. In still other embodiments, a hydrazine or semicarbazide can be used to form conjugates other than an oxime (Scheme 5), since reaction of a hydrazine or semicarbazide with the α-carbonyl group can provide the corresponding hydrazone or semicarbazone respectively. For this embodiment and the use of bis-aminoxy linkers to form oximes incorporating aminoxy functions for further protein and biologically active or biologically compatible agents linkage (see, e.g., PCT Publication No. WO2010/040147).

(26) In a general method, the species (13) is dissolved in a suitable solvent or buffer. A desired amount of the solution of (13) is incubated with a solution containing the carbonyl modifier for about 1-24 hours at a pH of about 3-11 and at a temperature of about 10-60° C. After the incubation is complete, the solution is subjected to centrifuge filtration. Removal of the excess reagents followed by optional purification provides the corresponding oxime or oxime analog product.

(27) Exemplary Bifunctional Reagents

(28) Exemplary bifunctional reagents that can be used to prepare the conjugates described herein are shown in Scheme 6, where Z can be, for example, a covalent bond, a C1-20 alkylene, 0, or a polyethylene glycol link, and representative X groups include Cl, Br, and I.

(29) ##STR00063##
For example, the reagents shown in Scheme 6 can be treated with, e.g., proteins P that include a nucleophilic group (e.g., a thiol group) to form homodimers (Scheme 7).

(30) ##STR00064##
Alternatively, conjugates can be prepared from, e.g., two different proteins (e.g., thiol-containing entities) using stepwise methodologies as shown in Scheme 8 with two differing nucleophilic reagents P and P′ as the second reaction is often much slower than the first. Any homodimer species formed can be separated from monomers, e.g., by centrifugation or HPLC.

(31) ##STR00065##

(32) The differential reactivity of α-halo acetophenone functional groups compared to the corresponding benzylic halides also can be exploited in the preparation of heterodimers and other conjugates (Scheme 9). For example, for the differentially activated halides shown below, the first protein moiety can be introduced selectively by displacement of the halide, alpha to the carbonyl, followed by reaction of the benzylic halide. This principle of exploiting differential reactivity by mixing groups of varying, but specific, thiol reactivity can be further extended to include halo-acetamides and the like, in conjunction with more reactive functions such as a-haloacetophenones.

(33) ##STR00066##

(34) It is to be noted that additional systems can be derived from all the foregoing containing ketone moieties by reduction of the carbonyls. Further, these synthetic schemes can be generalized to other biologically active agents or biologically compatible agents.

(35) Conjugates Including Alkyne and Azide Groups for Cycloaddition

(36) Yet another significant application of a-halobenzyl and a-halo-acetophenones is represented by the heterobifunctional substrates below containing azide or alkyne functionality (Scheme 10). These two functional groups can undergo a Huisgen 1,3-dipolar cycloaddition reaction, a transformation that is regarded as the exemplar of “click chemistry.” The facility with which this reaction proceeds, however, is dependent upon a number of factors that impact its rate. For example, the chemistry generally requires the addition of cupric salts to be practical. The use of cupric salts in conjunction with the synthesis of protein conjugates is somewhat limiting because of its potential toxicity, interferences with other metals bound by the native protein, and the difficulties associated with manufacturing. Catalysis of this reaction has also been described using the enzyme acetylcholinesterase as a template: when this enzyme is incubated with libraries of acetylenes and azides, cycloaddition is catalyzed by binding complementary entities in appropriate apposition for reaction.

(37) ##STR00067## ##STR00068##

(38) The halo and azide functionality can be independently processed in the production of heterodimers. A variety of macromolecules and/or drugs can be combined by first displacing the halide and then adding an alkyne-containing entity to the azide in a 1,3-dipolar addition, or vice versa. An alternative scheme in which both the halide-containing moiety and the alkyne are linked to the ring and the azide is borne by a separate molecule also provides similarly useful heterobifunctional adducts.

(39) The general utility of halomethyl ketones in pairings with cysteine nucleophiles can be exploited using ω-acetylenic halomethyl ketones (Scheme 11). The latter can then be condensed with various azide containing payloads in the above manner.

(40) ##STR00069##

(41) Reduced Conjugates

(42) The conjugated products of α-haloacetophenone substrates, or any other bifunctional reagent that includes a carbonyl group, can also be reduced to the corresponding alcohols (Scheme 12). Reducing agents such as sodium cyanoborohydride (NaBH.sub.3CN) are capable of quantitatively reducing the ketone. Chiral reduction of the ketone is also possible using methods and reagents known in the art. The reduction affords a new class of compounds incorporating a benzylic alcohol function that can be used in addition to, or in lieu of, their unsaturated precursors. Reduction eliminates the ketone as a point of attack by biological nucleophiles and enzymes and can afford a more chemically stable functional group. Indeed, we have found pegylated conjugates that include a benzylic alcohol moiety are more stable over a broader pH range than their unsaturated precursors. Accordingly, the increased stability may offer particular advantages in certain applications.

(43) ##STR00070##

(44) In keeping with the utility of benzylic type conjugates, we have investigated various benzylic halides as substrates for proteins containing free thiols, and found them to be excellent substrates. For example, we have prepared proteins conjugated to polyethylene glycol polymers using the same type of protocols employed for the α-thioacetophenone system. Although displacements of halides from benzylic halides are not as rapid as observed for the corresponding a-haloacetophenones, we have successfully prepared protein conjugates, site-specifically and quantitatively. Thus, we have produced yet another class of conjugates and conjugated products which do not possess the alcohol and ketone functions of the aforementioned systems. Conjugates and conjugated products derived from benzylic halides may provide special stability characteristics that can be employed to extend the duration of action of protein therapeutics.

(45) The scope of conjugated products available from halide-based substrates, incorporating the features above, can be further extended and applied to numerous applications.

(46) Benzoquinone and Related Conjugates

(47) Nucleophilic additions to 1,4-benzoquinones can occur rapidly under a variety of conditions including reactions in aqueous media, and a proposed mechanism for nucleophilic addition is shown in Scheme 13.

(48) ##STR00071##
In Scheme 13, “PSH” represents an exemplary protein that includes a nucleophilic thiol group that is reacted with 1,4-benzoquinone. The incipient product is represented as the 2-ene-1,4-dione (i), which is expected to aromatize to the hydroquinone (ii). In an oxidative environment, hydroquinone (ii) converts to the benzoquinone (iii) to complete the cycle. Thus, depending upon the redox potential and the oxidative or reductive environment, the product may exist primarily in either quinone or hydroquinone form.

(49) Further, the addition of amines (Yadav et al., Monatsh. Chem. 139, 1317-1320; Tandon et al., Teterahedron Letters 50:5896-5902, 2009); and Barbosa et al., Molecules 15, 5629-5643, 2010) and thiols (Katritzky et al. Synthesis: 777-787, 2008) to the parent benzoquinone occurs at modest temperature and must be controlled to avoid multiple additions to the ring. Facile reactions of simple amino acids independent of the side chain have also been reported (Morrison et al., Arch. Biochem. Biophys. 134:515-523, 1969), and it was stated that it was difficult to isolate appreciable quantities of the mono-substituted benzoquinone since the second mole equivalent added so rapidly. The 1,4-benzoquinone reaction of horse heart cytochrome c, which contains no free cysteines, was also reported: conjugate formation was attributed to protein primary amino groups or the phenolic groups of tyrosines.

(50) The synthetic methods described herein can also be employed with compounds (e.g., proteins) that include more than one nucleophilic group. For example, Scheme 14 shows a possible mode of reactivity when either a 1,4-benzoquinone reagent or an α-halo carbonyl reagent is combined with a compound (e.g., a protein, a biologically active agent, or a biologically compatible agent) that includes two nucleophilic groups. For example, the two nucleophilic groups can add to different carbons of a benzoquinone ring. When an α-halo carbonyl reagent is used, one nucleophilic moiety can displace the halo leaving group, and the second nucleophilic group can form, e.g., a tetrahedral adduct with the carbonyl group.

(51) ##STR00072##
These modes of reactivity are also available to compounds (e.g. proteins, biologically active agents, or biologically compatible agents) that include more than two nucleophilic group (e.g., three or four nucleophilic groups). For example, proteins, which contain diverse nucleophiles, successive intramolecular reactions between such nucleophiles and the quinone ring can occur as shown in Scheme 15.

(52) ##STR00073##
In this scheme, the protein PSH includes a thiol nucleophilic group as well as three other nucleophilic groups (Nuc.sub.1, Nuc.sub.2, and Nuc.sub.3). Thus, depending upon the reactivity of the substituted quinone and the reactivity of protein nucleophiles well-disposed for reaction in intermediate (iv), the quinone may ultimately be up to tetra-substituted by a single conjugated protein (as in intermediate (vii)). While 1,4-benzoquinone is used for exemplary purposes, other quinone reagents (e.g., substituted quinones or isomeric benzoquinones) can also be used in the methods.

(53) Accordingly, benzoquinone chemistry can serve as a useful strategy for preparing, e.g., commercially useful conjugates by judiciously controlling reaction conditions. The chemistry of 1,4-benzoquinones is described herein, and analogous methods can be used to prepare conjugates using 1,2- or 1,3-benzoquinones.

(54) Conjugates Via Thiol-Reactive 1,4-Benzoquinones

(55) 1,4-Benzoquinone systems can be exploited as site-specific labeling agents of proteins containing free thiols. The regiospecificity of attack may depend upon the pattern and extent of ring substitution. For example, with 2-bromo-1,4-benzoquinone (3) the primary ribosomal protein S4 from E. coli (S4 protein; Bellur et al., Nucleic Acids Res. April; 37(6):I 886-96, 2009), reacts predominantly by adding to the ring and eliminating bromine. By contrast, wild-type annexin V, which also possesses a lone free cysteine, predominantly reacts by adding to the ring without elimination of bromine Apparently, the pattern of cysteine reactivity of protein targets is dependent upon the steric accessibility of the protein thiol in its local environment. We have found that in certain instances, the free cysteine thiols of two separate protein molecules can be linked to a single 1,4-benzoquinone ring. In other instances, binary units (e.g., quinone rings, each bearing a protein), can be linked in series (Scheme 16, shown for one positional isomer). Accordingly, quinones can be used as scaffolds to conjugate thiol-containing proteins to produce, e.g., homo- or hetero-protein dimers, or to conjugate biologically active or biologically compatible agents to a target protein.

(56) ##STR00074##

(57) For example, the protein annexin V can be conjugated to the S4 protein, by first labeling annexin V with 1,4-benzoquinone and then treating the resulting conjugate with the S4 protein (Scheme 15). Similarly, we have prepared dimers of S4 thiol that are selectively conjugated by a single 1,4-benzoquinone ring (Scheme 16). Alternatively, a process to crosslink binary benzoquinone-protein units can be employed such that the proteins can reside on separate quinones (Scheme 17). Thus, based on the variable requirements of the quinone ring for conjugating specific entities, the appropriate strategies can be identified and employed to guide the most effective use of quinones as site-specific reagents for the conjugation of protein and biologically active or biologically compatible agents to proteins, and specifically, as mediators of protein dimer formation.

(58) The various scenarios we have developed for exploiting 1,4-benzoquinone reactivity in the production of protein conjugates bearing commercially viable protein and biologically active or biologically compatible agents are described in the figures below (Schemes 17-19).

(59) ##STR00075##

(60) ##STR00076##

(61) ##STR00077##

(62) Parent 1,4-Benzoquinone

(63) In the simple case of addition to the parent quinone without a leaving group substituent (Scheme 20, illustrated for conjugating a protein and biologically active or biologically compatible agents to a protein), there is no prospect for elimination of an atom or group by a displacement reaction under physiological conditions of temperature and pH, and the free cysteine adds to the quinone in Michael fashion to form a conjugate. Thus, the reactive quinone can be first conjugated to the protein target, thereby producing the hydroquinone, which is then oxidized to the quinone oxidation state. The oxidation can be accomplished either spontaneously in the presence of oxygen, or by the use of oxidizing reagents such as excess benzoquinone, CeSO.sub.4, potassium ferricyanide, cerium ammonium nitrate, or various other oxidizing agents known in the art to effect redox transformations. A second Michael addition to the ring with the desired protein or biologically active or biologically compatible agent is then implemented.

(64) ##STR00078##

(65) Alternatively, the parent system can be exploited by inverting the addition steps, as in Scheme 21, to first label quinones to carry a biologically active or biologically compatible agent (e.g., a polyethylene glycol polymer) as a ring substituent, which is then reacted with the target protein to produce the hydroquinone product.

(66) ##STR00079##

(67) As indicated above, we have discovered that 1,4-benzoquinone can be exploited as a site-specific labeling agent of several proteins containing free thiols, and can be used to crosslink protein and biologically active or biologically compatible agents to the target protein or to produce homo- or hetero-protein dimers. In certain instances, the free cysteine thiols of two separate protein molecules can be linked to a single 1,4-benzoquinone ring. As examples, homodimers of the S4 protein (S4-BQ-S4), and the heterodimers of S4 and annexin V, annexin V and annexin V-128, have been prepared site-specifically (e.g., to a single site on the protein framework), by attachment of the quinone to the free thiol of each protein component.

(68) Monosubstituted 1,4-Benzoquinones

(69) Monosubstituted quinones can also be used in any of the methods described herein. Exemplary monosubstituted quinones include haloquinones, alkylthioquinones, or alkylquinones. For instances in which the quinone is substituted with an atom or a group X that can function as a leaving group (Scheme 22) a scenario in which X is displaced to give the substituted quinone can obtain. This displacement allows for a second addition to the ring (with thiol bearing protein and biologically active or biologically compatible agents) without an oxidation step which can have advantages. Thus two successive additions to the ring can be effected resulting in attachment of the protein PSH to the quinone framework, followed by the addition of the second protein or biologically active or biologically compatible agent to the ring (or vice versa). Additionally, the use of certain monosubstituted benzoquinones (e.g., alkylquinones) can be used to block certain positions of the quinone ring from reacting with the nucleophilic group, thereby guiding the regiochemistry of the reaction sequence.

(70) ##STR00080##

(71) Disubstituted 1,4-Benzoquinones

(72) Disubstituted 1,4-benzoquinones can be useful substrates for introducing two entities onto a ring in tandem displacement reactions. Disubstituted 1,4-benzoquinones such as 2,6-dibromo-1,4-benzoquinones are available commercially. Similarly, dialkylquinones can also be used in the methods described herein. Tandem additions to the quinone are possible through the sequence described in Scheme 23.

(73) ##STR00081##

(74) It will be appreciated that the pattern of reactivity of the protein will dictate the sequence to be employed. It will also be appreciated by those trained in the art that, having the three possible options allows for flexibility in design of quinones carrying protein and biologically active or biologically compatible agents. For example, if the protein is not able to displace a potential leaving group, but able to add nucleophiles to the ring, then ring reactions can be accomplished through sequence A. Alternatively, if the protein is capable of displacing leaving group substituents, it can be directed toward particular positions of the ring.

(75) Protein Crosslinking: “One Ring, One Protein”

(76) We have established that 1,4-benzoquinones can act as highly specific and reactive labeling reagents of cysteine thiols of diverse proteins, e.g., annexin V, annexin V-128, and S4. The general nature of this reaction defines a path to protein dimers in stepwise fashion: (1) conjugation of the quinone to the protein, (2) oxidation of the product hydroquinone, and (3) addition of a second protein thiol. Whereas the formation of monoconjugates between protein and quinone substrates has proved to be general, the coupling reaction of the second protein could, in certain instances, be challenging. In instances where addition of a second protein to a quinone ring may be difficult to effect, an alternative strategy has been developed that is based on the specificity of binary 1,4-benzoquinone-protein units as thiol-protein acceptors. In this manner, protein dimers and multimers can be prepared. We have combined two such units to give dimers by employing bis-thiol coupling regents so that each ring need not bear more than one protein molecule (Scheme 22).

(77) For example, we have been able to achieve annexin V homodimers corresponding to (7) by condensing a first binary annexin V-1,4-benzoquinone unit (4) with the bis-thiol (5, X═S) to give the tethered system (6) (Scheme 17). Reaction of the latter with a second binary annexin V-1,4-benzoquinone unit results in the homodimer (7). This stepwise sequence, generalized in Scheme 3, can be adapted to the synthesis of heterodimers by choosing distinct protein and biologically active or biologically compatible agents RSH and R′SH. There are additional variants which achieve the objective of preparing bis-quinones bearing separate protein and biologically active or biologically compatible agents. that are shown in Scheme 24, illustrated for 1,4-benzoquinone. Eq. 1 utilizes the parent bis-quinone (8) as a substrate for coupling proteins (protein and biologically active or biologically compatible agents) to form dimers. Another variant, which employs the unsymmetrical bis-quinone (9), can accept a second protein or biologically active or biologically compatible agent to form a dimer.

(78) ##STR00082## ##STR00083##

(79) It will be appreciated that the ability to craft polyfunctional tethers, including dendrimers, affords diverse possibilities for preparing multimeric protein and biologically active or biologically compatible agents.

(80) Since we have observed the formation of binary quinone-protein units with a diverse group of 1,4-benzoquinone substrates including alkyl- and halo-substituted quinones, the use of such units represents a powerful approach for exploiting a one ring-one protein motif in the application of fusion proteins. For example, halo-quinones can be used to direct thiol-containing substrates to specific positions of the ring via displacement reactions of halide. Alkyl substituents can be used to block specific positions of the ring to facilitate the formation of homogeneous products and limit toxic mechanisms.

(81) Further, a variety of tethers can be envisaged which employ alkyl, alkylene oxides, peptide or peptoid chains to join two quinone rings. The nucleophiles that initiate attack on the quinone framework may be oxygen-, nitrogen- or sulfur-based (X═O,S,N). Since alcohols, amines and thiols are all capable of being introduced onto quinone rings by specific displacements of nucleofuges, there is the prospect of combining various imaging agents, drugs and the like, with quinone substrates.

(82) As shown in Scheme 25, bis-quinones in which alkyl chains are linked directly to the quinone ring can be prepared by a chemist of ordinary skill by methods known in the art: (Russell et al., J. Chem. Soc., Chem. Commun., 1987; Witiak et al. Med. Chem. 1989, 32, 1636-1642). Still other exemplary methods are described in (Dillmore et al., Langmuir 20(17):7223-31, 2004; Yeo et al., Langmuir 22(25):10816-20, 2006; and Yousaf et al., J. Am. Chem. Soc. 121, 4286-4287, 1999). These analogs may be especially useful in the formation of multimers as the carbon chain tether is not displaceable by nucleophiles.

(83) ##STR00084##

(84) Diels-Alder Reactions of Quinones

(85) Quinones offer another opportunity for conjugate formation as these are well-known Diels-Alder dienophiles. The mode of reactivity is illustrated in Scheme 26 for a protein and biologically active agents-bearing dienes. The Diels-Alder reaction can be exploited to crosslink protein and biologically active or biologically compatible agents to proteins either by derivatizing the diene or the 1,4-benzoquinone, with the protein and biologically active or biologically compatible agents to be conjugated. The Diels-Alder reaction constitutes an excellent version of “click chemistry” in the fashion of 1,3-dipolar addition reactions as the chemistry is orthogonal to protein chemistry and is accelerated by water (Michito et al., Science 312:251-254, 2006, and references therein) and various catalysts.

(86) ##STR00085##

(87) There are precedents for facile reactions of thio-substituted p-benzoquinone with dienes such as cyclopentadiene, and we have successfully performed novel Diels-Alder reactions of protein-1,4-benzoquinone conjugates to demonstrate the feasibility.

(88) It will also be appreciated that the reactions of thiol nucleophiles cited above, can be effected by amines (e.g., Katritzky et al., Synthesis 777-787, 2008). These entities constitute another opportunity to exploit the conjugation of proteins to protein and biologically active or biologically compatible agents where the protein and biologically active or biologically compatible agents is attached to an appropriate amine.

(89) An extraordinary feature of quinone reactions of thiol-containing species which possess nucleophiles in the vicinity of a reactive thiol, is the possibility of multiple attachments to the quinone acceptor (Scheme 13). The reaction of the second nucleophile gives cyclized product and provides a basis for additional stability of quinone conjugates and related conjugated products. This feature can be especially advantageous for instances where duration of action is essential, e.g., therapeutic proteins, antibody conjugates. We have shown for instance that 2-aminoethylmercaptan derivatives have a strong tendency to form six member rings and that certain proteins appear to undergo secondary reactions when conjugated to 1,4-benzoquinones (Scheme 27). This feature may be exploited in the design of mutant proteins by fusing suitably designed peptide moieties that contain cysteine and lysine in proper apposition for tandem quinone reactions.

(90) ##STR00086##

(91) Halopyruvate Reagents

(92) Halopyruvate reagents can also be used to prepare conjugates described herein. Halopyruvates have been primarily studied in enzyme reactions where they are usually used as mimics of substrates for pyruvate-processing enzymes. In some instances, pyruvates have proved to react selectively with protein thiols, but they have also demonstrated a proclivity to covalently label other nucleophilic amino acid side chains (e.g., Korotchkina et al., Arch Biochem Biophys, 369(2)277-87, 1999; Stamps et al., Biochemistry, 37(28):10195-202, 1998; Abeysinghe et al., J. Mol. Biol. 220(1):13-16, 1991; Huynh, Arch. Biochem. Biophys. 284(2):407-12, 1999; and Vlahos et al., J. Biol. Chem. 265(33):20384-9, 1990).

(93) Exemplary methods are described herein. For example, in Schemes 28-31, PSH can be a protein containing at least one free thiol such as natural proteins as annexin V, α-1-antiprotease, human sonic hedgehog N-terminal protein, oncostatin M, primary ribosomal protein S4, and proteins containing targetable free cysteines (e.g., annexins I, III, IV, V, VI and VIII). The present invention can also be applied to antibodies, minibodies, diabodies, affybodies and the like, and mutant proteins in which amino acid residues have been mutated to cysteines.

(94) In various embodiments, the thiol group of free cysteines of proteins can undergo an alkylation reaction with α-halo pyruvyl systems to form the corresponding thioether by displacement of the halide as shown in Scheme 28.

(95) ##STR00087##
In certain preferred embodiments, the thioether can be a β-thioether as part of an α-ketoamide (Y═N), α-keto acid (YW═OH), α-keto ester (Y═OR, R=alkyl or optionally substituted aryl or payload), or α-diketone network. In other preferred embodiments the thioether may be a β-thio-α-diketone. In still other important above embodiments, the thioether produced may be part of a network containing a heterocyclic moiety in Y or W.

(96) The pyruvate entity may be in the form of an ester or amide function that carries a desired protein, biologically active agent, or biologically compatible in Scheme 28. Site specific alkylation of proteins such as annexin V, α-anti-protease, or human sonic Hedgehog N-terminal protein and the aforementioned affords several additional possibilities for functionalizing the activated carbonyl group within the α-dicarbonyl linkage. In certain embodiments, the chemoselective conversion of the α-dicarbonyl species to oximes using aminoxy substrates can be carried out.

(97) In a general method, the pyruvate is dissolved in a suitable solvent or buffer. A desired amount of the pyruvate solution is incubated with a solution containing the carbonyl modifier for about 1-24 hours at a pH of about 3-11 and at a temperature of about 10-60° C. After the incubation is complete, the solution is subjected to centrifuge filtration. Removal of the excess reagents followed by optional purification provides the corresponding oxime or oxime analog product.

(98) As described herein for, e.g., conjugates derived from α-halocarbonyl and related functional groups, in some instances it may be desirable to convert the α-carbonyl of the protein conjugate to a hydroxy function by reduction using, e.g., sodium cyanoborohydride (Scheme 29).

(99) ##STR00088##

(100) Acrylic Acid Derivatives and their Cyclic Analogs

(101) In various embodiments, the thiol groups of free cysteines of proteins can undergo alkylation reactions with acrylic acid derivatives to form either the 3-propanoyl

(102) product when X is H, alkyl, or another non-displaceable group, or the β-thio-acryloyl adduct when X is a displaceable group such as bromine (Schemes 30 and 31). As shown in Scheme 29, in some embodiments, the Z group may also include a linker to a moiety R that is another protein, biologically active group, or biologically compatible group.

(103) ##STR00089##

(104) ##STR00090##

(105) In a general method, the protein is dissolved in a suitable solvent or buffer. A desired amount of the protein solution is incubated with the acrylic acid derivative for about 1-10 hours at a pH of about 6-10 and at a temperature of about 10-60° C. After the incubation is complete, the solution is subjected to centrifuge filtration. Removal of the excess reagents followed by optional purification provides the corresponding 3-thio substituted carbonyl systems.

(106) Peptides, Polypeptides and Proteins

(107) Proteins, peptides, and polypeptides may be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. Exemplary peptides, polypeptides, and proteins that can be used in the methods described herein are also described in, for example, U.S. Patent Publication No. 20100099649, which is herein incorporated by reference in its entirety. Still others include annexins, α-1-antiprotease, human sonic hedgehog N-terminal protein, oncostatin M, primary ribosomal protein S4, and, generally, other targetable free cysteines.

(108) Modified proteins can also be used in the methods described herein, where the native sequence or molecule is altered in such a way without materially altering the membrane binding affinity of the protein. Such modified proteins can be produced by chemical, genetic engineering, or recombinant techniques. The modification can include sequence modification through the addition of several amino acid residues, and/or an addition/deletion of an amino acid at a single site on the native or genetically engineered sequence. In the context of the present invention, modified proteins include proteins modified to include a cysteine residue having a free thiol (—SH) group.

(109) For example, a modified protein can have an amino acid sequence with at least one amino acid substitution (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 substitutions) compared to the naturally occurring sequence. The protein may contain, for example, 1 to 12, 1 to 10, 1 to 5, or 1 to 3 amino acid substitutions, for example, 1 to 10 (e.g., to 9, 8, 7, 6, 5, 4, 3, 2) amino acid substitutions. Alternatively, the modified protein has an amino acid sequence that has at least 35%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the amino acid sequence of the naturally occurring peptide.

(110) Biologically Active and Biologically Compatible Agents

(111) In addition to proteins, other biologically active agents and biologically compatible agents can be used in the methods described herein. Exemplary agents include polymers (e.g., polyethylene glycol), nucleic acids, carbohydrates (e.g., monosaccharides, disaccharides, oligosaccharides, and polysaccharides) such as polysialic acid, small molecule therapeutic agents, imaging agents, diagnostic agents, prophylactic agents, and optical agents. The resultant conjugates be used, e.g., for targeted delivery of the biologically active agent or the biologically active agents. Conjugates that include annexin proteins may be of particular interest. As shown herein in the Examples, the annexin conjugates prepared retain their biological activity post-modification. Further, the use of these conjugates (e.g., conjugates that include an annexin protein) as couriers to elevated sites of apoptosis/necrosis may be useful in methods of medical treatment and may be particularly important in the case of heart and cells of other vital organs. See, e.g., Kenis et al., “Annexin A5 uptake in ischemic myocardium: demonstration of reversible phosphatidylserine externalization and feasibility of radionuclide imaging,” J. Nucl. Med. 51(2):259-67, 2010.

(112) In addition to the use of the conjugates described herein for methods of selective delivery of therapeutic agents to sites of elevated apoptosis and necrosis. The preparation of fusion proteins is well known in the art (see, e.g., Chamow et al., Antibody Fusion Proteins, Wiley-Liss, 1999). For example, recombinant DNA methods can be used to prepare fusion proteins. In particular, fusion proteins that include annexin can be useful for targeted delivery of therapeutic agents. Exemplary fusion proteins that include annexin are described in, e.g., U.S. Pat. Nos. 7,407,475 and 7,262,167.

(113) The conjugates and fusion proteins can also include, in addition to an annexin protein, various proteins, biologically active agents, or biologically compatible agents, including those described in U.S. Pat. Nos. 7,906,118 and 7,534,585, herein incorporated by reference in its entirety. Exemplary groups can be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin, or a combination thereof.

(114) Exemplary cytokines include interleukins (IL), interferons (IFN), chemokines, tumor necrosis factor receptor ligands (e.g. 4-1BBL, OX40L, GITRL), killing inhibitor receptor (KIR) ligands, killing activatory receptor (KAR) ligands, IFN regulatory factors (IRFs) and B cell stimulatory factors. For example, the conjugates can include cytokines such as MIF (macrophage migration inhibitory factor), HMGB-1 (high mobility group box protein 1), TNF-α (tumor necrosis factor α), any of interleukins 1-19 and 23-24, any of chemokines 5, 10, 19, and 21, MCP-1 (monocyte chemotactic protein-1), any of macrophage inflammatory proteins1A and 1B, ENA-78, MCP-1 (monocyte chemoattractant protein), GRO-.beta., Eotaxin, interferon-α, interferon-β, interferon-γ, G-CSF, GM-CSF, SCF, PDGF, MSF, Flt-3 ligand, erythropoietin, thrombopoietin, CNTF, leptin, oncostatin M, VEGF, EGF, FGF, P1GF, insulin, hGH, calcitonin, Factor VIII, IGF, somatostatin, tissue plasminogen activator, and LIF.

(115) Anti-cancer antibodies can also be used in the conjugates or fusion proteins described herein. For example, anti-cancer antibodies include, but are not limited to, hR1 (anti-IGF-1R) hPAM4 (anti-MUC1), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEA), hMN-15 (anti-CEA), hRS7 (anti-EGP-1) and hMN-3 (anti-CEA)

(116) In particular, the use of fusion proteins and/or of conjugates that include annexin can be useful for the treatment or prevention of diseases characterized by increased necrotic or apoptotic activity in cells. For example, the conjugates and fusion proteins can be used for the treatment or prevention of cancer and other proliferative diseases, inflammation and inflammatory diseases (e.g., inflammatory bowel disease and rheumatoid arthritis) Crohn's disease, and diabetes.

(117) Cancers that may be treated according to the methods described herein include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's macroglobulinemia, multiple myeloma, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma).

(118) Proliferative diseases that may be treated according to the methods described herein include dyslasia, benign dysproliferative disorders, leukoplakia, Bowen's disease, keratoses, Farmer's Skin, solar cheilitis, solar keratosis, obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders (e.g., a disorder in which there is abnormal proliferation of cells of the lymphatic system), chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Still other proliferative diseases are described in U.S. Pat. Nos. 5,639,600 and 7,087,648, hereby incorporated by reference.

(119) Therapeutic Agents

(120) Exemplary classes of therapeutic agents include, but are not limited to carbohydrates, anti-microbials, antiproliferative agents, rapamycin macrolides, analgesics, anesthetics, antiangiogenic agents, vasoactive agents, anticoagulants, immunomodulators, cytotoxic agents, antiviral agents, antithrombotic drugs, such as terbrogel and ramatroban, antibodies, neurotransmitters, psychoactive drugs, oligonucleotides, proteins, lipids, and combinations thereof.

(121) Additional therapeutic agents that can be used in the methods described herein include, without limitation, growth hormone, for example human growth hormone, calcitonin, granulocyte macrophage colony stimulating factor (GMCSF), ciliary neurotrophic factor, and parathyroid hormone. Other specific therapeutic agents include parathyroid hormone-related peptide, somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl, norethisterone, clonidine, scopolomine, salicylate, salmeterol, formeterol, albeterol, valium, heparin, dermatan, ferrochrome A, erythropoetins, diethylstilbestrol, lupron, estrogen estradiol, androgen halotestin, 6-thioguanine, 6-mercaptopurine, zolodex, taxol, lisinopril/zestril, streptokinase, aminobutylric acid, hemostatic aminocaproic acid, parlodel, tacrine, potaba, adipex, memboral, phenobarbital, insulin, gamma globulin, azathioprine, papein, acetaminophen, ibuprofen, acetylsalicylic acid, epinephrine, flucloronide, oxycodone percoset, dalgan, phreniline butabital, procaine, novocain, morphine, oxycodone, aloxiprin, brofenac, ketoprofen, ketorolac, hemin, vitamin B-12, folic acid, magnesium salts, vitamine D, vitamin C, vitamin E, vitamin A, Vitamin U, vitamin L, vitamin K, pantothcnic acid, aminophenylbutyric acid, penicillin, acyclovir, oflaxacin, amoxicillin, tobramycin, retrovior, epivir, nevirapine, gentamycin, duracef, ablecet, butoxycaine, benoxinate, tropenzile, diponium salts, butaverine, apoatropine, feclemine, leiopyrrole, octamylamine, oxybutynin, albuterol, metaproterenol, beclomethasone dipropionate, triamcinolone acetamide, budesonide acetonide, ipratropium bromide, flunisolide, cromolyn sodium, ergotamine tartrate, and protein or peptide drugs such as TNF antagonists or interleukin antagonists. For example, the biologically active or biologically compatible agent can be an anti-inflammatory agent, such as an NSAID, corticosteriod, or COX-2 inhibitor, e.g., rofecoxib, celecoxib, valdecoxib, or lumiracoxib. The therapeutic agent may also include antibiotics.

(122) Diagnostic Agents

(123) Exemplary diagnostic agents which can be used in the methods described herein include, without limitation, imaging agents, such as those that are used in positron emission tomography (PET), computer assisted tomography (CAT), single photon emission computerized tomography, X-ray, fluoroscopy, and magnetic resonance imaging (MRI). Suitable materials for use as contrast agents in MRI include gadolinium chelates, as well as iron, magnesium, manganese, copper, and chromium chelates. Examples of materials useful for CAT and X-rays include iodine based materials. Other diagnostic agents that can be used in the methods described herein include those described in U.S. Patent Publication No. 20100099649, which is herein incorporated by reference in its entirety.

(124) Antibodies and Antibody Fragments

(125) Any of the conjugates described herein can include an antibody or an antibody fragment as described herein. The use of antibodies and antibody fragments in methods of treatments and in the preparation of immunoconjugates has been reviewed in: Holliger et al., Nature Biotechnology, 23(9):1126-1136 (2005); Wu et al., Nature Biotechnology 23(9):1137-1146 (2005); Tanaka et al., Cell Cycle 7(11):1568-1574 (2008); Kreitman, The AAPS Journal, 8(3):E532-E551 (2006); and Hudson et al., Nature Medicine, 9(1):129-132 (2003), each of which is hereby incorporated by reference.

(126) Antibodies include intact monoclonal and polyclonal antibodies, as well as various genetically engineered antibodies.

(127) Antibody fragments can be produced using standard methods. Exemplary antibody fragments include monovalent (e.g., Fab, scFv, single variable V.sub.H domain, and single variable V.sub.L domain fragments) and divalent fragments (e.g., Fab′.sub.2 fragments, diabodies, triabodies, tetrabodies, and minibodies). Truncated versions of monoclonal antibodies, for example, can be produced by recombinant methods in which plasmids are generated that express the desired monoclonal antibody fragment(s) in a suitable host. Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methods for preparing single polypeptide chain antibodies. Ward et al., Nature 341:544-546, 1989, describes the preparation of heavy chain variable domain which have high antigen-binding affinities. McCafferty et al. (Nature 348:552-554, 1990) show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage binds specifically to antigen, and that rare phage (one in a million) can be isolated after affinity chromatography. Boss et al. (U.S. Pat. No. 4,816,397) describes various methods for producing immunoglobulins, and immunologically functional fragments thereof, that include at least the variable domains of the heavy and light chains in a single host cell. Cabilly et al. (U.S. Pat. No. 4,816,567) describes methods for preparing chimeric antibodies.

(128) Immunoconjugates

(129) The invention also provides for the preparation of immunoconjugates and the use of these compounds, or compositions thereof, in methods of medical treatment. For example, any of the proteins described herein can further include an intact antibody or fragment, a single-chain variable fragment (scFv), a diabody, a minibody, or a scFv-Fc fragment. The use of immunoconjugates allows for the targeted delivery of a therapeutic to particular cells (e.g., tumor cells; see, e.g., Mak et al., Primer to the Immune Response, page 277, Academic Press, 2008). Annexin conjugates have also been studied for these applications (see, e.g., Backer et al., “Adapter protein for site-specific conjugation of payloads for targeted drug delivery,” Bioconjug. Chem. 15(5):1021-9, 2004, and Tanaka et al., “Preparation and characterization of a disulfide-linked bioconjugate of annexin V with the B-chain of urokinase: an improved fibrinolytic agent targeted to phospholipid-containing thrombi,” Biochemistry, 35(3):922-9, 1996). Accordingly, the preparation and study of cytokine immunoconjugates (e.g., cytokines conjugated to annexin) is of great interest for developing new therapeutic methods. For example, a tumor-targeting antibody-(Interferon α) conjugate has been prepared and studied for efficacy in the treatment of lymphoma (Rossi et al., Blood, 114:3864-3871, 2009). Accordingly, such strategies may be applied to the present multimeric proteins (e.g., dimeric cytokines) for the targeted delivery of these compounds to a cell.

EXAMPLES

(130) The following examples are meant to indicate the intrinsic specificity of the labeling components in the aforementioned specifications. A person trained in the art will appreciate the range of these examples demonstrating specificity on cysteine thiols such as annexin Cys-315, which, hitherto, have been challenging to site-specifically label.

(131) In all the examples described herein, annexin V and annexin V-128 retain their biological activity when modified at their respective single cysteines. For example, the Ca.sup.2+ dependent binding abilities of the modified proteins for 1:1 DOPC:DOPS liposomes (dioleoylphosphatidyl serine=DOPS, dioleoylphosphatidylcholine=DOPC) are not different from the native protein itself.

(132) Mass Spectrometry

(133) Samples for mass spectrometry analysis were prepared using Varian OMIX C4 Ziptips and eluted into 90% acetonitrile, 10% water, with 0.1% formic acid.

(134) For ESI-Mass spec, the samples were infused to a LCQ classic (Thermo) with a flow rate of 10 μL/min. Experimental conditions were as follows: spray voltage at 3 KV and capillary voltage and temperature at 47V and 250 degrees C., respectively. The data were collected continuously for 4 minutes and then were deconvoluted and analyzed by software ProMass (Thermo).

(135) For MALDI, the samples were mixed with 10 mg/ml alpha-cyano-4-hydroxy-cinnamic acid matrix (1:1 volume) before being spotted on a MALDI plate using the dry droplet technique. The samples were analyzed using a Bruker Daltonics Ultraflex TOF/TOF with linear mode and a laser power at 40-50%. Instrument settings were as follows: IS1 at 25.04 kV, IS2 at 23.21 kV, lens at 6.81 kV, and pulsed ion extraction at 18 ns. 500-1000 shots were typically collected for a spectrum. The data were analyzed using software FlexAnalysis by Broker Daltonics.

Example 1

Site-Specific Labeling of Recombinant Human Alpha-1 Antitrypsin or E. coli 30S Ribosomal Protein S4

(136) Recombinant human alpha-1 antitrypsin or E. coli 30S ribosomal protein S4 (1-5 micromolar) was treated with 1 mM 3-bromopyruvate in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After 5 minutes, the protein had completely reacted site-specifically as indicated by the shift of the parent mass to a single peak from 44,440 to 44,S26 (alpha-I), or from 23,338 to 23,424 (S4 protein).

Example 2

Site-Specific Labeling of Alpha-1 Antitrypsin or S4 Protein

(137) alpha-1 Antitrypsin or S4 protein (1-5 micromolar) was treated with 1 mM 3-bromoacetophenone in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After five minutes, the protein had completely site-specifically reacted as indicated by the shift of the parent mass at 44,440 to a single peak at 44,SS8 (alpha-I), or from 23,338 to 23,456 (S4 protein).

Example 3

Site-Specific Labeling of Human Annexin V

(138) Human annexin V (1-5 micromolar) was treated with 10 mM 3-bromo acetophenone in 100 mM triethanolamine, pH 8.0, at 25° C. After 20 minutes, the protein had completely reacted site-specifically as indicated by the shift of the parent mass at 35,805 to a single peak at 35,923.

Example 4

Site-Specific Labeling of Human Annexin V by 1,4-Benzoquinone

(139) Wild type human annex in V (1-5 micromolar) was treated with 100 micromolar 1,4-benzoquinone in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After five minutes the protein had completely reacted site-specifically as indicated by the shift of the parent mass at 35,805 to 35,913 (the combined mass of parent annexin and 1,4.benzoquinone.

Example 5

Site-Specific Labeling of the Primary Ribosomal Protein 84 from E. coli

(140) Ribosomal protein 84 from E. coli (1-5 micromolar) was treated with 100 micromolar 1,4-benzoquinone in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After five minutes, the protein had completely reacted as indicated by the shift of the parent mass at 23,338, to 23,446, (the combined mass of parent Ribosomal protein S4 and 1,4-benzoquinone).

Example 6

Site-Specific Labeling of Recombinant Human Alpha-1 Antitrypsin or E. coli 30S Ribosomal Protein S4

(141) Recombinant human alpha-1 antitrypsin or E. coli 30S ribosomal protein S4 (1-5 micromolar) was treated with 1 mM 3-bromopyruvate in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After 5 minutes, the protein had completely reacted site-specifically as indicated by the shift of the parent mass to a single peak from 44,440 to 44,S26 (alpha-I), or from 23,338 to 23,424 (S4 protein).

Example 7

Site-Specific Labeling of S4 Protein

(142) S4 protein (1-5 micromolar) was treated with 10 mM 3-bromocyclohex-en-1-one in 100 mM phosphate buffered saline at pH8.0 at 25° C. After 5 min, the protein had completely reacted site specifically as indicated by the shift of the parent mass at 23,338 to a single peak at 23,432.

Example 8

Conjugation of Human Annexin V and the Primary Ribosomal Protein S4 from E. coli by 1,4-Benzoquinone

(143) Wild type human annexin V (1-5 micromolar) was treated with 100-1000 micromolar 1,4-benzoquinone in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After five minutes, the protein had completely reacted site-specifically as indicated by the shift of the parent mass at 35,805 to 35,913 (the combined mass of parent annexin and 1,4-benzoquinone). The binary product was purified through centrifuge filtration and used freshly for subsequent reactions described below.

(144) The annexin-benzoquinone was then incubated with 1 molar equivalent of purified E. coli 84 protein (free of TCEP, b-ME etc) in pH 8.0 PBS at room temperature for 16 hours. The mass spectrum shows an almost quantitative reaction towards an annexin-84 heterodimer conjugated by a benzoquinone moeity.

Example 9

Conjugation of the Primary Ribosomal Protein 84 from E. coli by 1,4-Benzoquinone to Form Homodimers

(145) Ribosomal protein 84 from E. coli (1-5 micromolar) was treated with 100 micromolar 1,4-benzoquinone in 100 mM phosphate buffered saline at pH 8.0 at 25° C. After five minutes, the protein had completely reacted as indicated by the shift of the parent mass at 23,338, to 23,446 (the combined mass of parent Ribosomal protein 84 and 1,4-benzoquinone). The binary product was purified through centrifuge filtration and used freshly for subsequent reactions described below.

(146) The ribosomal protein 84-benzoquinone was then incubated with 1 molar equivalent of purified E. coli 84 protein (free of TCEP, b-ME etc) in pH8.0 PBS at room temp for two hours. The mass spectrum shows an almost quantitative reaction towards an 84 homodimer conjugated by a benzoquinone moeity.

Example 10

Crosslinking of Annexin V-I,4-Benzoquinone Units by 2,2′-(ethylenedioxy)-diethanethiol

(147) Annexin V was first reacted with 1 mM benzoquinone in pH8 PBS buffer at room temp for 5 minutes. This produced a nearly homogeneous product of annexin-benzoquinone as described in Example 7. After centrifuge filtration, the annexin-benzoquinone was further reacted with 10 mM compound of 2,2′-(ethylenedioxy)-diethanethiol in pH8 PBS buffer at room temp for 30 minutes. The mass spectrum suggests a homogeneous product in which one equivalent of 2,2′-(ethylenedioxy)-diethanethiol is added to the annexin-benzoquinone unit. After centrifuge filtration, the tethered product was incubated with one molar equivalent of annexin-benzoquinone in pH8 PBS at room temp overnight. The mass spectrum shows a peak corresponding to the annexin homodimer formed through linkage of annexin-benzoquinone units. The conversion is estimated to be greater than 30%.

Example 11

Reaction of 1,4-Benzoquinones with Cysteine Thiols of Proteins

(148) Site-Specific Formation of the Heterodimer Conjugate of Annexin V and Annexin V-128

(149) 10 μM Annexin V was reacted with 1 mM 1,4-benzoquinone in pH 8.0 phosphate buffer at room temperature for 5 minutes. The reaction leads to the complete conversion of annexin V, which becomes fused to 1,4-benzoquinone specifically via a thiol-group (annexin-S-benzoquinone, annexin-S-BQ) as detected by LCQ-MS. After separation of the small molecule fraction by centrifuge filtration, annexin-S-BQ was then incubated with an equimolar amount of annexin V-128, which produces annexin-V conjugated by benzoquinone to annexinV-128 thiol-specifically. The yield of the heteroconjugate is approximately 50%, as shown by SDS-PAGE and LCQ-MS.

(150) Site-Specific Formation of the Heterodimer Conjugate of Annexin V & E. coli Ribosomal Protein S4 Mediated by 1,4-Benzoquinone

(151) The reaction between E. coli ribosomal protein S4 and annexin V was carried out as above. A stable product is obtained in which the S4 protein and annexin V are both thiol-specifically linked to benzoquinone to give the conjugated product as above. The yield is greater than 50%, based on analysis by SDS-PAGE and LCQ-MS.

(152) Site-Specific Formation of the Conjugated Heterodimer of Annexin V & α1-Antiprotease Mediated by 1,4-Benzoquinone

(153) The reaction between α1-antiprotease and annexin V was carried out as above. A stable product is obtained in which α1-antiprotease and annexin V are both thiol-specifically linked to benzoquinone to give the conjugated product as above.

(154) Site-Specific Formation of the Homodimer of Annexin V Conjugated by 2,2′-Ethylenedioxy)diethanethiol

(155) 10 μM annexin-S-BQ is reacted with 1 mM HS—(CH.sub.2).sub.2O(CH.sub.2).sub.2O(CH.sub.2).sub.2SH (2,2′-ethylenedioxy)diethanethiol, HS-PEG2-SH)) compound in pH 8.0 phosphate buffer at room temperature for 1 hour. This leads to quantitative formation of an annexin-S-BQ-S-PEG2-SH adduct, as detected by LCQ-MS. After separation of the small molecule fraction using centrifuge filtration, annexin-S-BQ-S-PEG2-SH was incubated with an equimolar amount of annexin-S-BQ in pH8.0 phosphate buffer at room temp overnight. A homodimer corresponding to annexin-S-BQ-S-PEG2-S-BQ-S-annexin is produced in 40-60% yield (based on SDS-PAGE and LCQ-MS analyses).

(156) Site-Specific Formation of the Heterodimer of Annexin V and Annexin V-128 Conjugated by 2,2′-Ethylenedioxy)diethanethiol

(157) 10 μM Annexin-S-BQ-S-PEG2-SH is reacted with 1 mM benzoquinone in pH 8.0 phosphate buffer at room temperature for 5 minutes. This leads to a complete conversion to an annexin-S-BQ-S-PEG2-S-BQ product as detected by LCQ-MS. After separation of the small molecule fraction using centrifuge filtration, the protein product was then incubated with an equimolar amount of annexin V-128 in pH 8.0 phosphate buffer at room temperature overnight. The reaction with annexin V-128 forms the conjugated product corresponding to annexin-S-BQ-S-PEG2-S-BQ-S-annexin-V-128: in this product, each annexin protein is thiol-specifically linked to their respective benzoquinone rings.

(158) Site-Specific Formation of the Heterodimer of Annexin V and α1-Antiprotease, Conjugated by 2,2′-Ethylenedioxy)diethanethiol

(159) 10 μM annexin-S-BQ-S-PEG2-SH is reacted with 1 mM benzoquinone in pH8.0 phosphate buffer at room temperature for 5 minutes. This leads to complete conversion to an annexin-S-BQ-S-PEG2-S-BQ product based on analysis by LCQ-MS. After separation of the small molecule fraction using centrifuge filtration, the protein product is then incubated with an equimolar amount of alpha-1 antitrypsin. The reaction with alpha-1 antitrypsin produces annexin-S-BQ-S-PEG2-S-BQ-alpha-antiprotease as indicated by SDS-PAGE.

(160) Site-Specific Thiolation of 1,4-Benzoquinone Leading to Proteins Conjugated to PEG Polymers

(161) 10 μM annexin-BQ is treated with a five molar excess of PEG-SH (20 KDa) in pH8.0 phosphate buffer at room temperature overnight. A product corresponding to annexin-V-BQ-S-PEG with a yield approximating 50-60% as estimated by SDS-PAGE is obtained.

(162) 1 mM PEG-SH (20 KDa) is treated with a 10× molar excess of benzoquinone in pH8.0 phosphate buffer at room temperature for 5 minutes to yield a product corresponding to PEG-S-BQ. After separation of the small molecule fraction using centrifuge filtration, 10-20 μM PEG-S-BQ is further reacted with an equimolar amount of annexin V-128 or alpha-1 antitrypsin in pH8.0 phosphate buffer at room temperature overnight. The reaction leads to the pegylated products of PEG-S-BQ-annexin-V 128 or PEG-S-BQ-alpha-1 antiprotease with yields approximating 30-40%, as detected by SDS-PAGE.

Example 12

Site-Specific Crosslinking of PEGNH2 (20K) to Alpha-1 Antiprotease via 4-Bromomethylbenzoic acid

(163) 10 μM alpha-1 antiprotease is incubated with a 5× molar excess of 4-bromomethyl-benzoylamide of PEGNH.sub.2 (20 KDa) [prepared from 4-bromomethylbenzoic acid and ethyl chloroformate, which is then added in excess to PEGNH.sub.2 20K and stirred overnight] in pH8.0 phosphate buffer at room temperature overnight. The reaction with alpha-1 antiprotease yields 4-alpha-1-S-benzyl-PEGamide, as detected by SDS-PAGE.

Example 13

Site-Specific Crosslinking of PEGNH2 (20K) to Annexin V-128 Via 4-Bromoacetylbenzoic acid

(164) 10 μM annexin V-128 is treated with a 5× molar excess of 3- or 4-bromoacetylbenzoyl-PEGamide (prepared from 3- or 4-bromoacetylbenzoic acid and ethyl chloroformate, which is then added in excess to PEGNH.sub.2-20K and stirred overnight) in pH 8.0 phosphate buffer at room temperature. SDS-PAGE and MALDI mass spectrometry indicated the formation of the conjugated pegylated product.

Example 14

Synthesis and Study of an Annexin V/IL-10 Conjugate (Scheme 32)

(165) Annexin V or annexin V-128 (concentration varied from several μg/ml to several mg/ml) was incubated with 10 mM PLP in 25 mM PBS (pH 6.5) at 37° C. for 4-16 hours. After the incubation, the sample was subjected to centrifuge filtration to remove excess PLP from solution and then analyzed by mass spectrometry. Various aminoxy substrates, which form oximes with carbonyl species, were used to distinguish the N-terminal keto-amide product of the annexin protein from that of the parent and to demonstrate that the product was obtained in excellent yield.

(166) The resulting annexin ketoamide was then incubated with 100 mM bis-1,6-aminoxy hexane at room temperature for 4 to 16 hours. The reaction mixture was constantly shaken. After terminating the reaction, the sample was subjected to centrifuge filtration to remove the excess bis-aminoxy compound from solution. The 6-aminoxy-oxime of annexin V or V-128 was thus obtained.

(167) ##STR00091##

(168) AKT-IL-10 Ketoamide

(169) IL-10 was cloned and expressed with the tripeptide N-terminal extension alanine-lysine-threonine (AKT) to give AKT-IL-10. (Witus et al., J Am Chem Soc. 132:16812-7, 2010). The AKT-IL-10 was then treated with PLP as above for 16 hours; the small molecule fraction was then separated, and the N-terminal keto amide of AKT-IL-10 was thus obtained.

(170) Reaction Between the N-Terminal Ketoamide of AKT-IL-10 and 6-Aminoxyannexin

(171) 6-Aminoxy-annexin, obtained as described above, was incubated with 30 micromolar N-terminal ketoamide of AKT-IL-10 at room temperature overnight, at pH 6.5 in 25 mM PBS. The structure of the crosslinked product of the annexin protein and IL-10 (Scheme 33) was established by mass spectrometry analysis of the enzymatically digested heterodimer.

(172) ##STR00092##

(173) Crosslinking of 6-Aminoxy-Annexin and 6-Aminoxy-AKT-IL-10 to Form the Conjugate

(174) Aminoxy annexin V oxime was incubated with 1 mM terephthaldehyde at room temperature for 10 min (buffer, pH). The reaction to form the mono-aldoxime with the protein species is complete within 10 minutes. Only one of the two aldehyde groups of the substrate is consumed. The annexin-aldehyde (was then treated with the 6-aminoxy-AKT-IL-10. Using conditions described herein, the 6-aminoxy-oxime of AKT-IL-10 was then condensed with the annexin mono-aldoxime to give the terephthaldehyde-linked conjugate (Scheme 34).

(175) ##STR00093##

(176) Evaluation of the Efficacy of Annexin:IL-10 Conjugates in an Arthritis Animal Model.

(177) The annexin:IL-10 conjugates A and B of Scheme 35 were each evaluated for their therapeutic efficacy in chronic inflammatory arthritis in mice by injecting them into joints. TNF transgenic (TNF-Tg) mice were used as a model of chronic inflammatory arthritis. Either conjugate A or conjugate B was injected into joints of TNF-Tg mice. MRI and lymphatic imaging were used during the 4-months following injection to assess changes in synovial volume and lymph flow from joint tissues to local draining lymph nodes. Joint inflammation, bone erosion, and cartilage loss were examined by histologic analyses. Lymphatic vessel formation was assessed using immunohistochemistry. Intra-articular administration of either conjugate A or conjugate B significantly attenuated the increase in synovial volume and increased lymphatic vessel number in joint sections compared to IL-10 during the 4-month period. This was accompanied by reduced inflammation area, bone erosion, cartilage loss, and osteoclast numbers. Lymph flow from joints to local draining lymph nodes was slower in TNF-Tg mice than in wild-type littermates and was significantly improved with either conjugate A or conjugate B as compared with treatment by IL-10. Conjugate A or conjugate B also exhibited greater duration of action and less systemic toxicity than IL-10.

(178) ##STR00094##

Example 15

Reaction of 1,3-Cyclopentadiene with the 1:1 Conjugate of Annexin V and 1,4-Benzoquinone

(179) Cyclopentadiene was freshly prepared and dissolved in methanol to a final concentration of 100 mM. Annexin V-1,4-benzoquinone, prepared according to the methods described herein, was also prepared to a 20 μM final concentration in 25 mM phosphate buffer (pH=6.5).

(180) Annexin V-1,4-benzoquinone (100 μL) was then mixed with cyclopentadiene (20 μL) at 4° C. for 20 minutes. Analysis by mass spectroscopy confirmed that the Diels-Alder adduct was formed. Annexin V under the same conditions does not react with cyclopentadiene.

Other Embodiments

(181) All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.

(182) While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

(183) Other embodiments are within the claims.