WO2006115800A2

WO2006115800A2 - Enhanced wound healing utilizing an anti-her2 antibody coupled to a tnf alpha

Info

Publication number: WO2006115800A2
Application number: PCT/US2006/013815
Authority: WO
Inventors: Tzu-Hsuan Huang; Sherie L. Morrison
Original assignee: The Regents Of The University Of California
Priority date: 2005-04-15
Filing date: 2006-04-12
Publication date: 2006-11-02
Also published as: WO2006115800A3

Abstract

This invention provides novel compositions and methods to enhance wound healing and/or to reduce scar formation. In certain embodiments the methods comprise contacting the wounded tissue with a composition comprising an anti-HER2/neu antibody attached to a TNF-α.

Description

ENHANCED WOUND HEALING UTILIZING AN ANTI-HER2 ANTIBODY COUPLED TO A TNFALPHA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of and priority to USSN 60/671 ,707, filed

April 15, 2005, which is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This work was supported in part by grant CA87990 from the National

Institutes of Health. The Government of the United States of America has certain rights in this invention.

FIELD OF THE INVENTION

[0003] This invention pertains to the field of wound healing. In particular, this invention pertains to the discovery that an anti-HER2-TNFα construct is effective in enhancing wound healing in mammals.

BACKGROUND OF THE INVENTION

[0004] The ΗER2/neu (c-erhB-2) proto-oncogene encodes a transmembrane protein tyrosine kinase growth factor receptor, pl85^HER2 (Aldyama et al. (1986) Science 232:1644- 1646) with extensive homology to the human epidermal growth factor (EGF) receptors (Coussens et al. (1985) Science 230:1132-1139). The intrinsic tyrosine kinase activity of ΗERllneu has been shown to trigger a network of signaling pathways, culminating in responses including cell division, differentiation, and proliferation. Abundant evidence has supported the role of this proto-oncogene in tumori genesis. It has been demonstrated that overexpression of ΗER2/neu correlates with the poor prognosis in breast and ovarian cancer patients due to the increased metastatic potential and resistance to anti-cancer therapeutics of the malignant cells (Slamon et al. (1987) Science 235:177-182; Hung et al. (1992) Cancer Lett 61:95-103; Yu et α/.(1998) MoI Cell 2:581-591; Hudziak et al. (1988) Proc. Natl. Acad. ScI, USA, U.S.A. 85: 5102-5106; Lichtenstein et al. (1990) Cancer Res 50: 7364-7370). In addition it has recently been demonstrated that ΗERllneu plays an essential role in the repair of injured airway (Vermeer et al. (2003) Nature 422:322-326) and corneal epithelia (Xu et al (2004) Invest Ophthalmol Vis Sd ASAlIlAlS¹,?,). ΗERllneu is expressed on the basolateral surface of epithelial cells, and injury allows its ligand, heregulin, to interact with ΗER2/neu, promoting cell proliferation and repair of the epithelial layer.

[0005] The effects induced by the tyrosine ldnase activity of HER2/neu are mediated by an intricate downstream signaling cascade. It has been shown that homodimers of ΗER2/neu activate phosphatidylinositol 3-kinase (PI3K) (Hu et al. (1992) MoI Cell Biol 12:981-990). Activation of PI3K generates PtdIns-3,4-P2, which in turn recruits and activates Akt (Zhou et al (2000) J. Biol. Chem. 275:8027-8031). Activated Akt phosphorylates specific targets such as pro-caspase-9 (Cardone et al. (1998) Science 282:1318-1321) and Bad (del Peso et al. (1997 Science 278:687-689), promoting cell survival. In addition, activated ΗERl/neu has been shown to associate with a SH2 domain- containing protein, SHC (Meyer et al. (1994) MoI Cell Biol 14:3253-3262), leading to the activation of mitogen-activated protein ldnase (MAPK). Activated MAPK translocates to the nucleus and activates transcription factors thereby promoting cell growth and development (Lenormand et al. (1998) J Cell Biol 142:625-633). Thus the Akt and MAPK signaling pathways play an essential role in the cell survival and proliferation induced by ΗER2/neu activation. [0006] Antibodies targeting ΗERl/neu have been used for therapy of ΗER2/neu overexpressing tumors. Herceptin is a human IgGl recombinant antibody designed to block ΗER2/neu. Although Herceptin has been shown to exhibit a transient and modest agonistic effect in ΗER2/neu activation (Scott (1991) J. Biol. Chem. 266:14300-14305), it inhibits long term growth of ΗERl/neu overexpressing breast cancer cells in vitro (Hudziak et al. (1989) MoI. Cell Biol, 9: 1165-1172), and, in combination with taxanes, improves the survival of patients with HER2-positive metastatic breast cancer (Leyland- Jones et al.(lWΪ) Ann Oncol 12 Suppl 1: S43-47). Although the mechanism of the anti-proliferative effect of Herceptin remains unclear, preventing ligand binding to ΗERl/neu is believed to contribute to this effect. [0007] Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine secreted primarily by activated macrophages and monocytes. TNF-α exhibits a wide spectrum of biological activities including promoting cytolysis of some tumor cell lines by activating apoptosis (Laster et al. (1988) J Immunol 141:2629-2634, enhancing the anti-tumor effect of dendritic cells (Candido et al. (2001) Cancer Res 61:228-236) and activating host immunity against tumors (Hock et al. (1993) Proc Natl Acad Sd USA 90:2774-2778). Therefore, TNF-α could be a promising anti-cancer therapeutic. However, clinical use of TNF-α as an anticancer drug is hampered by its severe systemic toxicity.

SUMMARY OF THE INVENTION

[0008] This invention pertains to the surprising discovery that a construct comprising tumor necrosis factor alpha (TNFα) attached to an anti-HER2/neu antibody shows efficacy in promoting wound healing and/or reducing scar formation.

[0009] Thus, in certain embodiments this invention provides a composition for enhancing wound healing in a mammal, where the composition comprises an anti- ΗER2/neu antibody attached to a tumor necrosis factor alpha (TNF-α). In certain embodiments the antibody is a single chain antibody (e.g., a single chain Fv antibody (scFv)). In various embodiments the heavy chain of the antibody is a fusion protein with TNF-α, and the light chain of the antibody is covalently linked to the heavy chain. In various embodiments the light chain of the antibody is a fusion protein with TNF-α, and the heavy chain of the antibody is covalently linked to the heavy chain. In certain embodiments the covalent linkage between the heavy and light chain of the antibody is via a disulfide linkage. In certain embodiments the TNF-α and the antibody comprising the fusion protein are joined directly or by a peptide linker (e.g., (Gly₄Ser)₃ (SEQ ID NO:5)) linker. In certain embodiments the antibody is a C6 antibody or a herceptin antibody. In various embodiments the antibody comprises C6MH3-B1 variable heavy (VH) region and/or a C6MH3-B1 variable light (VL) region. In certain embodiments the antibody is C6MH3-B1 scFv. The antibody can be joined to the TNF-α directly or by a linker. In certain embodiments the antibody is joined to the TNF-α by peptide linker (e.g. a (Gly₄Ser)₃ (SEQ ID NO:5)). In various embodiments the antibody joined to the TNF-α forms a single chain fusion protein. In certain embodiments the TNF-α is a human TNF-α. The TNF-α can be a full length native TNF-α, or it can be truncated, mutated, or otherwise modified (e.g. via conservative substitutions) and it can have reduced or eliminated TNF-α activity, but retain the ability to bind to two other TNF-α molecules. In certain embodiments the TNF-α is a human TNF-α comprising the mutation Y87S. In certain embodiments the TNF-α is a murine TNF-α {e.g., a murine TNF-α comprising the mutation S 147Y). In certain embodiments the anti-HER2/rce« antibody attached to a tumor necrosis factor alpha (TNF- α) is a fusion protein comprising a C6MH3-B1 scFv attached (directly or via a linker) to a /human TNF-α comprising the mutation Y87S. In certain embodiments the composition comprises a complex consisting of three anti-HER2-TNFα.

[0010] In certain embodiments the anti-HER2/ne« antibody attached to a tumor necrosis factor alpha (TNF-α) is present in a pharmacologically acceptable excipient. In various embodiments the excipient is suitable for topical administration to the skin or eye. In certain embodiments the composition is in a unit dosage formulation.

[0011] Also provided are methods of enhancing wound healing in a mammal. The methods typically involve contacting a wounded tissue in the mammal with a composition as described above in a dosage sufficient to enhance wound healing. In certain embodiments the wound is selected from the group consisting of an acute wound, a chronic wound, a surgical wound, and an optical wound. In certain embodiments the wound is selected from the group consisting of a wound to the skin, a wound to a mucosal surface, and a wound to an internal tissue or organ.

[0012] This invention also provides dressings {e.g., surgical dressings, bandages, etc.) where the dressing is impregnated with a composition as described above. In certain embodiments the dressing is a sterile dressing.

[0013] In various embodiments this invention provides a method of activating a

HER2 receptor. The method typically involves contacting the HER2 receptor with a trimerized anti-HER2 antibody. In certain embodiments the trimerized anti-HER2 antibody comprises a composition as described above.

[0014] Similarly, this invention also provides a method of increasing Rac induced cell migration. The method typically involves contacting a tissue in a mammal with a trimerized anti-HER2 antibody. In certain embodiments the trimerized anti-HER2 antibody comprises a composition as described above. the method comprising contacting a tissue in a mammal with a trimerized anti-HER2 antibody.

[0015] This invention also provides a composition comprising a first moiety attached to a first TNF-α, a second moiety attached to a second TNF-α, and a third moiety attached to a third TNF-α, where the first, second and third TNF-α interact to form a trimer thereby coupling the first, second, and third moieties, and the first, second and third moieties are independently selected from the group consisting of an antibody, a ligand, an epitope tag, a cytokine, a growth factor, a receptor, a cytotoxin, a detectable label, a lipid, and a liposome. [0016] Methods are also provided for forming a trimeric complex of a first moiety, a second moiety, and a third moiety. The methods typically involve providing the first moiety attached to a first TNF-α, the second moiety attached to a second TNF-α, and the third moiety attached to a third TNF-α; contacting the first, second, and third TNF-α with each other whereby the first, second and third TNFα interact to form a trimer thereby coupling the first, second, and third moieties to each other.

[0017] Methods are provided for enhancing wound healing. The methods typically involve contacting a wounded tissue with a polyvalent construct that specifically binds three or more HER2/neu receptors.

[0018] Similarly compositions are provided that comprise at least three ΗER2/neu specific antibodies. The ΗERl/neu specific antibodies can be the same or different antibodies and, in certain embodiments, they can be covalently or non-covalently joined together.

[0019] Kits for the enhancement of wound healing are provided herein. The kits typically comprise a container containing a composition as described above. In certain embodiments the composition is provided in a dressing (e.g., a sterile dressing) for a wound. In certain embodiments the container is an aersolizer for topical delivery to a wound. In certain embodiments the composition is formulated as a cream, lotion, salve, ointment, gel, and the like for topical administration. The kit can optionally include instructional materials teaching the use of the composition to enhance wound healing and/or to reduce the formation of scan tissue and/or adhesions. DEFINITIONS

[0020] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The term also includes variants on the traditional peptide linkage joining the amino acids making up the polypeptide. Preferred "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-terminus) refers to the free α-amino group on an amino acid at the amino terminal of a peptide or to the α-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide. Similarly, the term "carboxy terminus" refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide. Peptides also include essentially any polyamino acid including, but not limited to peptide mimetics such as amino acids joined by an ether as opposed to an amide bond. [0021] As used herein, an "antibody" refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0022] A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V_L) and variable heavy chain (VH) refer to these light and heavy chains respectively.

[0023] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'_2> a dimer of Fab which itself is a light chain joined to V_R-C_HI by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region thereby converting the (Fab')₂ dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region {see, Fundamental Immunology, W.E. Paul, ed., Raven Press, N. Y. (1993), for a more detailed description of other antibody fragments). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such Fab' fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes whole antibodies, antibody fragments either produced by the modification of whole antibodies or synthesized de novo using recombinant DNA methodologies. Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), more preferably single chain Fv antibodies (scFv) in which a variable heavy and a variable light chain are joined together (directly or through a peptide linker) to form a continuous polypeptide. In various embodiments the single chain Fv antibody comprises covalently linked V_H and V_L domains that can, for example, be expressed from a nucleic acid including V_H- and V_L- sequences either joined directly or through a peptide linker (see, e.g., Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883). While in certain embodiments, the V_H and VL are connected to each as a single polypeptide chain (directly or through a linker), in various embodiments the V_H and V_L domains can be associated non-covalently or covalently (e.g. through a disulfide linkage). The first functional antibody molecules to be expressed on the surface of filamentous phage were single-chain Fv's (scFv). Alternative expression strategies, however, have also been successful. For example Fab molecules can be displayed on phage if one of the chains (heavy or light) is fused to g3 capsid protein and the complementary chain exported to the periplasm as a soluble molecule. The two chains can be encoded on the same or on different replicons; the point is that the two antibody chains in each Fab molecule assemble post-translationally and the dimer is incorporated into the phage particle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S. Patent No: 5,733,743). Other useful expression systems include, but are not limited to and yeast display libraries. The recombinant expression of scFv antibodies and a number of other mechanisms for converting naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778). Particularly preferred antibodies should include all that have been displayed on phage and/or yeast (e.g., scFv, Fv, Fab and disulfide linked Fv (Reiter et al. (1995) Protein Eng. 8: 1323-1331), and also include bivalent, trivalent, quadravalent, and generally polyvalent antibody complexes.

[0024] With respect to antibodies of the invention, the term "immunologically specific" "specifically binds" refers to antibodies that bind to one or more epitopes of a protein of interest (e.g., ΗER2lneύ), but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.

[0025] The term "TNF-α" refers to full-length tumor necrosis alpha. The TNF-α can be from essentially any mammalian species. In certain preferred embodiments, the TNF-α is from a species selected from the group consisting of human, equine, a bovine, rodent, porcine, lagomorph, a feline, a canine, a murine, a caprine, an ovine, a non-human primate, and the like. TNF-α also includes truncated and/or mutated TNF-α. Mutated

TNF-α comprises one or more amino acid substitutions, insertions, and/or deletions, e.g., as described herein.

[0026] An anti-HER2/new antibody is an antibody that specifically binds a

ΗERllneu receptor. [0027] As used herein, the term "subject" refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, goat, rabbit, mouse, rat, or monkey.

[0028] The term "C6 antibody", as used herein refers to antibodies derived from

C6.5 whose sequence is expressly provided, for example, in U.S. Patents 6,512,097 and 5,977,322, and in PCT Publication WO 97/00271. C6 antibodies preferably have a binding affinity of about 1.6 x 10^" or better for ΗER2/neu. In certain embodiments C6 antibodies are derived by screening (for affinity to c-erbB-2 / ΗER2/neu) a phage display library in which a known C6 variable heavy (V_H) chain is expressed in combination with a multiplicity of variable light (V_L) chains or conversely a known C6 variable light chain is expressed in combination with a multiplicity of variable heavy (V_H) chains. C6 antibodies also include those antibodies produced by the introduction of mutations into the variable heavy or variable light complementarity determining regions (CDRl, CDR2 or CDR3), e.g., as described in U.S. Patents 6,512,097 and 5,977,322, and in PCT Publication WO 97/00271. In addition, C6 antibodies include those antibodies produced by any combination of these modification methods as applied to C6.5 and its derivatives.

[0029] A single chain Fv ("sFv" or "scFv") polypeptide is a peptide comprising a variable heavy (V_H) and a variable light (V_L) domain or equivalents, covalently linked together, directly or through, e.g. a peptide linker. The single chain Fv can, in certain embodiments be expressed from a nucleic acid including V_H- and V_L- encoding sequences either joined directly or joined by a peptide-encoding linker (see, e.g., Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883). The recombinant expression of scFv antibodies and a number of other mechanisms for converting naturally aggregated, but chemically separated light and heavy polypeptide chains from an antibody V region into a molecule that folds into a three dimensional structure substantially similar to the structure of an antigen-binding site are known to those of skill in the art (see e.g., U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778).

[0030] The phrase "reduction in scar formation" as used herein refers to the production of a scar smaller in size than would ordinarily have occurred in the absence of the active components and/or a reduction in the size of an existing scar. [0031] The term "conservative substitution" is used herein to refer to replacement of amino acids in a protein with different amino acids that do not substantially change the functional properties of the protein. Thus, for example, a polar amino acid might be substituted for a polar amino acid, a non-polar amino acid for a non-polar amino acid, and so forth. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows a schematic representation of the anti-HER2/πe« ScFv-TNF- α . TNF-α (light circle) was fused to the carboxy terminus of the ScFv (C6MH3-B1) antibody by a NWSHPQFEK streptavidin tag (filled rectangle ) and (Gly₄Ser)₃ (SEQ E) NO: 5) linker (dark circles). A trimeric structure was formed when three monomers interact through the TNF-α moieties.

[0033] Figures 2 A, 2B, and 2C illustrate the production and characterization of anti- HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv- TNF-α(S 147Y). Figure 2A: SDS-

PAGE analysis under reducing conditions of purified recombinant TNF-α and ScFv-TNF-α fusion protein visualized by Coomassie blue staining. Lane 1: recombinant murine TNF-α( rTNF-α), Lane 2: TNF-α fused to the anti-HER2/rce« ScFv (anti-HER2/neu ScFv-TNF-α), Lane 3: TNF-α(S147Y) fused to the anti-HER2/?zew ScFv (anti-HER2/ne« ScFv-TNF- αS147Y), Lane 4: TNF-α fused to an anti-dansyl ScFv (anti-dansyl ScFv-TNF-α). Figure 2B: Cross-linking assay of ScFv-TNF-α fusion proteins. Anti-HER2/neu ScFv-TNF-α untreated (lane 1) or treated with the crosslinker EGS (ethylene glycolbis) (lane 2) and anti- HER2/neu ScFv-TNF-α(S147Y) untreated (lane 3) or treated with EGS (lane 4) were subjected to SDS-PAGE followed by electroblotting to a nitrocellulose membrane. The strips were reacted with biotinylated anti-TNF-α and detected with horseradish peroxidase- conjugated strepavidin. Figure 2C: FPLC analysis under non-denaturing conditions. Anti- HER2/neu ScFv-TNF-α and anti-BDER2/neu ScFv-TNF-α(S147Y) were analyzed by FPLC. For comparison BSA (67kDa) and Miles IgG (15OkDa) separated under identical conditions are shown. Fraction size is ImI. [0034] Figures 3 A and 3B illustrate antigen binding and cytotoxic activity of the anti-HER2/neu ScFv-TNF-α fusion proteins. Figure 3A: Anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S147Y) bind ΗER2/neu. D2F2/E2, a murine mammary cell line expressing high levels of human HER2/neu, was reacted with (1) anti-HER2/neu ScFv- TNF-α, (2) anti-HER2/neu ScFv-TNF-α(S147Y), or (3) anti-dansyl ScFv- TNF-α. Cells were then washed and incubated sequentially with biotinylated rat ani-mouse TNF-α and PE-labeled streptavidin. Dashed lines represent signal from cells without addition of recombinant protein. Figure 3B: The cytotoxic activity of recombinant TNF-α and different ScFv-TNF-α fusion proteins against the murine L929 fibroblast cell line in the presence of Actinomycin D. After incubation for 24h with increasing doses of different proteins, viable cells were stained with crystal violet dye that was then dissolved with methanol to allow colorimetric evaluation. The experiment was performed three times in triplicate; error bars correspond to the SD of the measurement.

[0035] Figures 4A and 4B show that anti-HER2/neκ ScFv-TNF-α and anti-

ΗER2/neu ScFv-TNF-α(S147Y) inhibit apoptosis induced by Actinomycin D through ΗERllneu binding. Figure 4A: The anti-apoptotic activity of anti-HER2/new ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S 147Y) was determined using the human SKBR3 breast cancer cell line. After incubation for 24h with increasing doses of the different proteins in the presence of 4 μg/ml of Actinomycin D, MTS solution was added to the viable cells, and the plates were measured on a ELISA reader at 490 nm. Figure 4B: The anti-apoptotic effect of anti-HER2/rce« ScFv-TNF-α and anti-HER2/ne« ScFv-TNF-α(S147Y) requires ΗERllneu binding. SKBR3 cells were incubated with 25 nM of either anti-HER2/rceu

ScFv-TNF-α or anti-HER2/neu ScFv-TNF-α(S147Y) and increasing concentrations of anti- HER2 IgG3 or anti-dansyl IgG3 in the presence of 4 μg/ml of Actinomycin D. MTS solution was added, and the plates were measured on an ELISA reader at 490 nm. The experiments in Figures 4A and 4B were performed three times in triplicate; error bars indicate the SD of the measurements.

[0036] Figures 5A and 5B show that anti-HER2/rceu ScFv-TNF-α (Figure 5A) and anti-HER2/ft<?M ScFv-TNF-α(S147Y) (Figure 5B) induce tyrosine phosphorylation of HER2/πew. 1.5 x 10⁶ SKBR3 cells were treated for 5 min with different concentrations of the fusion proteins as well as the culture medium for SKBR3 cells (medium) and CHO medium, a concentrated culture medium from the non-transfected CHO cell line, Pro-5, which was prepared using the same protocol used for the production of the anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S147Y) proteins. The cell lysates were separated by SDS-PAGE followed by electroblotting to PVDF microporous membranes. The strips were reacted with 4G10, a monoclonal mouse anti-phosphotyrosine antibody and the bound 4G10 was detected with horseradish peroxidase-conjugated second-step reagents. Blots that had been probed for the phosphorylated proteins were stripped and reprobed with a rabbit polyclonal antibody against HER2/neu.

[0037] Figure 6 shows that anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-

TNF-α(S 147Y) induced robust activation of p44/42 MAPK (ERK1+2). SKBR3 cells (1.2 x 10⁶) were treated with 50 nM concentrations of the different anti-BER2/neu proteins for the indicated times. The cell lysates were separated by SDS-PAGE gel followed by electroblotting to PVDF microporous membranes. The strips were reacted with a monoclonal mouse anti-phosphop44/42 MAPK and the bound antibody was detected with horseradish peroxidase-conjugated second-step reagents. To confirm equal loading of protein samples, blots that had been probed for the phosphorylated proteins were stripped and reprobed with a rabbit polyclonal antibody against MAPK. The intensity of anti- phosphop44/42 MAPK was normalized with the intensity of anti-MAPK for each indicated time point, and the values obtained were divided by the value at time 0 to obtain the fold activation for p44/42 MAPK. [0038] Figure 7 shows that anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-

TNF-α(S 147Y) induced activation of Akt. SKBR3 cells (1.2 x 10⁶) were treated with 50 nM of the different anti-HER2//7ew proteins for the indicated times. The cell lysates were separated by SDS-PAGE followed by electroblotting to PVDF microporous membranes. The strips were reacted with a polyclonal rabbit anti-phosphoAkt and the bound antibody was detected with horseradish peroxidase-conjugated second-step reagents. To confirm equal loading of protein samples, blots that had been probed for the phosphorylated proteins were stripped and reprobed with a rabbit polyclonal antibody against Akt. The intensity of anti-phosphoAkt was normalized with the intensity of anti-Akt for each indicated time point, and the values obtained were divided by the value at time 0 to obtain the fold activation for Akt.

[0039] Figure 8 shows that activation of both MAPK and Akt contribute to the anti- apoptotic effect induced by anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF- α(S147-Y). 5 x 10⁴ SKBR3 cells were treated with 100 nM of either anti-HER2/neu ScFv- TNF-α or anti-HER2/neu ScFv-TNF-α(S147Y) alone or with the indicated concentration of the MAPK inhibitor, U0126, and/or the Akt inhibitor, LY294002, in the presence of 4 μg/ml of Actinomycin D (ACD) for 24 hour. MTS solution was then added to the viable cells, and the plates were measured on a ELISA reader at 490 nm. These experiments were performed three times in triplicate; error bars correspond to the SD of the measurement.

[0040] Figure 9 shows that anu-BER2/neu ScFv-TNF-α and anti-HER2/new ScFv-

TNF-α(S 147Y) facilitate the repair of mechanically injured epithelia. Mechanically injured Caco2 colonic epithelial cells were treated with either CHO medium (line 1), anti-

ΗER2/neu ScFv-TNF-α 100 nM (line 2), anti-HER2/new ScFv-TNF-α(S147Y) 100 nM (line 3), PBS (line 4), anti-HER2/πeκ IgG3 with the C6MH3-B1 variable region 100 nM (line 5), anti-HER2/7zeM IgG3 with the Herceptin variable region 100 nM (line 6). The injured epithelial monolayers were photographed using phase contrast microscopy at the indicated times; wound edges are highlighted for clarity.

[0041] Figure 10, panels A-D, show that enhancement of wound healing involved the binding of the trimeric anti-HER2/rc<?« ScFv antibodies to ΗERl/neu on injured epithelial cells. Mechanically injured Caco2 colonic epithelial cells were treated with either anti-HER2/Wu ScFv-TNF-α 100 nM (panel A, lines 1-3), anti-HER2/ne« ScFv-TNF-α 100 nM + soluble ΗER2/neu 20OnM (panel B, lines 1-3), anti-HER2/new ScFv-TNF-α(S147Y) 100 nM (panel C, lines 1-3), or anti-HER2/neκ ScFv-TNF-α(S 147Y)IOO nM + soluble ΗERl/neu 20OnM (panel D, lines 1-3). The injured epithelial monolayers were photographed using phase contrast microscopy for the indicated times; wound edges are highlighted for clarity.

DETAILED DESCRIPTION

[0042] This invention pertains to the surprising discovery that a construct comprising tumor necrosis factor alpha (TNFα) attached to an anti-HER2/new antibody shows efficacy in promoting wound healing and/or reducing scar formation.

[0043] It is envisioned, that the compositions described herein will find significant use in treating wounds, especially in the treatment of chronic wounds or wounds that are resistant to healing, such as those seen bedridden patients and/or in patients with diabetes mellitus. In addition, it is contemplated that such compositions and methods will also find use in surgical settings to promote the healing related to surgical incisions, and in the opthamalogical setting to promote healing of eye injuries (e.g., corneal scrapes and the like). [0044] As used herein, the term "wound" is used to describe skin wounds are treated by the formulations and the methods described herein as well as wounds to various mucosal surfaces {e.g., oral and nasal mucosa, etc.), and various tissue and/or organ wounds. A skin wound is a break in the continuity of skin tissue. Skin wounds are generally characterized by several classes including punctures, incisions, including those produced by surgical procedures, excisions, lacerations, abrasions, atrophic skin, or necrotic wounds and burns.

[0045] Chronic wounds are a frequently encountered problem in elderly and bedfast patients and are typically produced by trauma or pathologic insult. Characteristics of chronic wounds include, but are not limited to, a loss of skin or underlying tissue. Chronic wounds typically do not heal with conventional treatment. The edges of chronic wounds unlike other types of wounds are not approximated and are typically characterized by an accompanying tissue deficit.

[0046] Tissue wounds include wounds to an internal organ, such as a blood vessel, intestine, colon, etc. The materials of the invention are useful for enhancing the wound healing process in tissue wounds whether they arise naturally or as the result of surgery.. For instance, during the repair of arteries the vessel needs to be sealed and wound healing must be promoted as quickly as possible. The compositions of the invention can speed up that process.

[0047] The methods for promoting/enhancing wound healing can be accomplished by applying the compositions described herein to the wound. In some embodiments, the uptake of the biologically active component(s), can be enhanced using the application of an electric field. The electric field aids in the delivery of the biologically active component through the skin or material that has begun to form the scar. This method helps to continue the administration of the biologically active component even after the skin has begun to regenerate to repair the wound. This embodiment may be accomplished using electrophoresis and/or electroosmosis. Electrophoresis operates by having an electrode with the same charge as that of the ionic molecules above the solution adjacent to the skin which is the site of administration. The ions will be repelled and migrate through the skin and/or other tissue into the lower parts of the wound. Electroosmosis involves the use of a negative electrode causing an electric current to flow resulting in the movement of the biological active agents. One system for accomplishing this is described in U.S. Pat. Nos: 6,129,696, 4528265; 5,503,632; and 6,129,696, and the like, and in PCT Publication WO 00/47273, and the like.

[0048] It was also a surprising discovery that TNF-α can be used to readily form trimeric entities even when the TNF-α is coupled to another moiety. Thus, in certain embodiments, this invention provides general methods of forming trimers/trimeric complexes and uses for those trimers and/or complexes thus formed. Typically this is accomplished by providing a first moiety attached to a first TNF-α, a second moiety attached to a second TNF-α, and a third moiety attached to a third TNF-α. The first and/or second and/or third moiety can be the same or different moieties.

II. Anti-HER2/we«-TNFα Chimeric Moieties.

[0049] As explained above, it was a surprising discovery that chimeric moieties comprising an anti-HER2/n<?w antibody attached to a native (wildtype) or modified TNF-α can be effectively used to enhance wound healing in a number of contexts. In certain embodiments the anti-ΗER2/neu antibodies are chemically conjugated to the anti-TNF-α, however in certain preferred embodiments, the anti-HER2/πew antibody are expressed as a fusion protein with the TNF-α. When produced as a fusion protein the antibody component can be directly fused to the TNF-α or attached by means of a peptide linker (e.g., a (G₄S)₃ linker (SEQ ID NO:5).

A) Anti-HER2/neu antibodies. [0050] A number of anti-HER2/ne« antibodies are know to those of skill in the art and are well suited for use in the methods and compositions of this invention. While the antibody can be from essentially any mammalian species, to reduce immunogenicity, it is desirable to use an antibody that is of the species in which the wound healing composition is to be used. In other word, for use in a human, it is desirable to use a human, humanized, or chimeric human antibody.

[0051] Fully human anti-HER2/new antibodies are well known to those of skill in the art. Such antibodies include, but are not limited to the C6 antibodies such as C6.5, DPL5, G98A, C6MH3-B1, B1D2, C6VLB, C6VLD, C6VLE, C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5, C6MH3-D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, C6MH3-C3, C6MH3-B9, C6MH3-B5, C6MH3-B48, C6MH3-B47, C6MH3-B46, C6MH3-B43, C6MH3-B41, C6MH3-B39, C6MH3-B34, C6MH3-B33, C6MH3-B31, C6MH3-B27, C6MH3-B25, C6MH3-B21, C6MH3-B20, C6MH3-B2, C6MH3-B16, C6MH3-B15, C6MH3-B11, C6MH3-B1, C6MH3-A3, C6MH3-A2, and C6ML3-9. These and other anti- ΗER2/neu antibodies are described in U.S. Patents 6,512,097 and 5,977,322, in PCT Publication WO 97/00271, in Schier et al. (1996) JMoI Biol 255: 28-43, Schier et al. (1996) JMoI Biol 263: 551-567, and the like.

[0052] As described in U.S. Patents 6,512,097 and 5,977,322 other anti-HER2/«eu antibodies can readily be produced by shuffling light and/or heavy chains followed by one or more rounds of affinity selection. Thus in certain embodiments, this invention contemplates the use of one, two, or three CDRs in the VL and/or VH region that are CDRs described in the above-identified publications.

[0053] The invention need not be limited to the use of these anti-HER2/?7ew antibodies and other such antibodies as they are know to those of skill in the art can be used in the compositions and methods described herein.

B) TNF-α and modified TNF-α

[0054] In various embodiments chimeric moieties of this invention comprise a TNF- α joined to the anti-HER2/new antibody. The TNF-α can be a full length wildtype TNF-α, a TNF-α fragment, and/or a mutated TNF-α. Typically the TNF-α fragment and/or the mutated TNF-α is a TNF-α that forms a trimer with two other copies of the same modified TNFα fragment.

[0055] It was discovered that TNF-α activity is not required for the wound healing activity to be present in the anti-HER2/new~TNFα constructs. Thus, in certain embodiments, the use of a modified TNF-α having reduced or eliminated TNF-α activity is contemplated.

[0056] While mutations/modifications that reduce endogenous TNF-α activity are desired in certain embodiments, it is also desirable that the modified TNF-α still retain the ability to form trimers with two other wild-type or modified TNF-α molecules. [0057] Means of identifying such modified TNF-α molecules are routine to those of skill in the art. In one illustrative approach, a library of truncated and/or mutated TNF-α is produced and screened for TNF-α activity and for trimer formation. Methods of producing libraries of polypeptide variants are well known to those of skill in the art. Thus, for example error-prone PCR can be used to create a library of mutant and/or truncated TNF-α (see, e.g., U.S. Patent 6,365,408).

[0058] The resulting library members can then be screened according to standard methods know to those of skill in the art. Thus, for example, TNF-α activity can be assayed by quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity (see, e.g., Decker and Lohmann-Matthes (1988) J. Immunol. Meth.,25;l 15(l):61-9), by active cytotoxicity on a target cell line, e.g., L929 cells (see e.g., Fiemu et al. (1996) J. Trauma-Injury Infection & Crit. Care., 40(4): 564-567, and the like).

[0059] Similarly, assays for protein/protein interactions are well known to those of skill in the art and include, but are note limited to gel-shift assays, fluorescence resonance energy transfer (FRET) systems, and the like.

[0060] In certain embodiments, site-directed mutagenesis is used to introduce specific mutations to inactivate the TNF-α (e.g., a Y87S mutation in a human TNF-α), and the like.

[0061] These methods are intended to be illustrative and not limiting. Using the teaching provided herein, other suitable modified TNF-αs can readily be identified and produced.

C. Attachment of the anti-HER2/»eM antibody to the TNF-α.

[0062] Generally speaking, the anti-HER2/πeu antibody and the TNF-α can be joined together in any order. Thus, for example, the anti-HER2/πeu antibody can be joined to either the amino or carboxy terminal of the TNFα. The antibody can also be joined to an internal region of the TNF-α, or conversely, the TNF-α can be joined to an internal location of the antibody , as long as the attachment does not interfere with binding of the antibody to the ΗERllneu receptor or the interaction of the chimieric moieties to for multimeric (e.g. trimeric aggregates) via interaction of the TNF-α. [0063] The anti-HER2/neκ antibody and the TNF-α can be attached by any of a number of means well known to those of skill in the art. In certain embodiments, the TNF- α is conjugated, either directly or through a linker (spacer), to the antibody. In certain embodiments, however, it is preferable to recombinantly express the chimeric moiety as a fusion protein.

a) Chemical conjugation of the anti-HER2/rae« antibody to the TNF-α.

[0064] In certain embodiments, the anti-HER2/neu antibody (e.g., C6.5, C6MH3-

Bl, G98A, ML3-9, H3B1, B1D2, etc.) is chemically conjugated to the TNF-α molecule. Means of chemically conjugating molecules are well known to those of skill.

[0065] The procedure for conjugating two molecules varies according to the chemical structure of the agent. Polypeptides typically contain variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH₂) groups, which are available for reaction with a suitable functional group on the other peptide, or on a linker to join the molecules thereto.

[0066] Alternatively, the antibody and/or the TNF-α can be derivatized to expose or attach additional reactive functional groups. The derivatization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford Illinois. [0067] A "linker", as used herein, typically refers to a molecule that is used to join the antibody to the TNF-α. In various embodiments, the linker is capable of forming covalent bonds to both the antibody and to the TNF-α. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In certain embodiments, the linker(s) can be joined to the constituent amino acids of the antibody and/or the TNF-α through their side groups (e.g., through a disulfide linkage to cysteine). In certain preferred embodiments, the linkers are joined to the alpha carbon amino and/or carboxyl groups of the terminal amino acids of the antibody and/or the TNF-α. [0068] A bifunctional linker having one functional group reactive with a group on the antibody and another group reactive on the TNF-α, can be used to form the desired conjugate. Alternatively, derivatization can involve chemical treatment of the targeting moiety. Procedures for generation of, for example, free sulfhydryl groups on polypeptides, such as antibodies or antibody fragments, are known (See U.S. Patent No: 4,659,839).

[0069] Many procedures and linker molecules for attachment of various compounds including radionuclide metal chelates, toxins and drugs to proteins such as antibodies are known. See, for example, European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075. In particular, production of various immunotoxins is well-known within the art and can be found, for example in "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190 (1982); Waldmann (1991) Science, 252: 1657; U.S. Patent Nos. 4,545,985 and 4,894,443, and the like.

b) Production of fusion proteins.

[0070] In certain embodiments, chimeric anti-HER2-TNF-α fusion proteins of the present invention are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.

[0071] DNA encoding the fusion proteins {e.g. anti-HER2/πew-TNF-α) of this invention can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett, 22: 1859-1862); the solid support method of U.S. Patent No. 4,458,066, and the like.

[0072] Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences.

[0073] Alternatively, subsequences can be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments can then be ligated to produce the desired DNA sequence.

[0074] In certain embodiments, DNA encoding fusion proteins of the present invention can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the gene for TNF-α is PCR amplified, using a sense primer containing the restriction site for, e.g., Ndel and an antisense primer containing the restriction site for HindIIL This can produce a nucleic acid encoding the mature TNF-α sequence and having terminal restriction sites. An antibody having "complementary" restriction sites can similarly be cloned and then ligated to the TNF-α and/or to a linker attached to the TNF-α. Ligation of the nucleic acid sequences and insertion into a vector produces a vector encoding TNF-α joined to the anti-HER2/new antibody. [0075] While the two molecules can be directly joined together, one of skill will appreciate that the molecules can be separated by a peptide spacer consisting of one or more amino acids. Generally the spacer will have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. In certain embodiments, however, the constituent amino acids of the spacer can be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.

[0076] The nucleic acid sequences encoding the fusion proteins can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene is typically operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences. [0077] The plasmids of the invention can be transferred into the chosen host cell by well-known methods such as electroporation, calcium chloride transformation for E. coli and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.

[0078] Once expressed, the recombinant fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N. Y.: Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N. Y., and the like).

Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.

[0079] One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the anti-HΕR2/new-TNF-α fusion protein may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (see, e.g., Debinski et al. (1993) J. Biol. Chein., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205: 263-270). Debinski et al., for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine. [0080] The cloning and expression of an anti-HER2/ne« antibody ([* ]) is illustrated herein in Example 2. As shown in Example 2, the heavy and light chain variable regions of an anti-HER2/neu antibody (e.g., C6MH3-B1) can be amplified by PCR and using the appropriate restriction sites, inserted into the human γ3 heavy chain (pAH480) and K light chain (pAG4622) expression vectors respectively (Coloma et al. (1992) J Immunol Methods 152: 89- 104). To construct the anti-human ΗER2/neu ScFv (C6MH3-B 1 )-TNF-α fusion protein, overlap PCR can be used to introduce a sequence optionally encoding a streptavidin tag and/or peptide linker upstream of the TNF-α gene with the forward primer.

[0081] The product can then be used as template for a second PCR using the same reverse primer and a second forward primer. The final PCR product can be ligated into, e.g., the TA vector. The vector can be digested with EcoRV and Avrll to release the DNA fragment containing the TNF-α gene which is inserted into, e.g., a TA vector containing the antibody (e.g., C6MH3-B1) gene in a position 3' of the antibody gene. The resulting plasmid can be digested with EcoRV and BamHl and the fragment containing C6MH3-B1 ScFv joined to TNF-α can then be inserted into a vector (e.g., a pcDNA3.1 vector (Invitrogen)). The resulting plasmid, contains the coding region for anύ-ΗER2/neu antibody ScFv followed by a streptavidin tag, a peptide linker linker, and a TNF-α.

[0082] One of skill would recognize that modifications can be made to the anti-

HER2/ne«-TNA-α fusion proteins without diminishing their activity/efficacy. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons.

D. Other Multi-valent anti-HER2/ne« moieties. [0083] In certain embodiments this invention contemplates the use of multi-valent, preferably trivalent, quadravalent, pentavalent or greater anti-HER2/πeu moieties to enhance wound healing and/or to reduce scar formation.

[0084] Multivalent anti-HER2/rcew. moieties can be produced by any of a number of methods. For example, linkers having three, four, or more reactive sites can be reacted with anti-HER2/πeM antibodies to form a trimer or greater conjugate.

[0085] In certain embodiments, phage display, bacterial display, or other display systems can be used to express and display multiple copies (e,g., at least 3, at least 4, at least 5, at least 6 copies, etc.) of an anti-HER2//ieu antibody and thereby effectively provide a multivalent anti-HER2/72eu moiety. IH. Formation of other trimeric moieties.

[0086] It was a surprising discovery that TNF-α can be used to readily form trimeric entities even when the TNF-α is coupled to another moiety. Thus, in certain embodiments, this invention provides general methods of forming trimers/trimeric complexes and uses for those trimers and/or complexes thus formed. Typically this is accomplished by providing a first moiety attached to a first TNF-α, a second moiety attached to a second TNF-α, and a third moiety attached to a third TNF-α. The first and/or second and/or third moiety can be the same or different moieties.

[0087] Essentially any moiety that it is desired to incorporate into such a complex can be utilized in this method. Typically the moieties will be selected so that they do not interfere with the TNF-α interactions that give rise to the formation of a trimer. In certain embodiments the moieties can be attached to the respective TNF-α with flexible linkers to reduce the likelihood of steric hinderance. Suitable moieties include, but are not limited to cytokines, growth factors, antibodies, ligands, receptors, detectable labels, cytotoxins, lipid complexes, liposomes, drug encapsulation vehicles, and the like.

[0088] The TNF-α can be native (wildtype) or modified TNF-α having endogenous

TNF-α activity or not, e.g. as described above. In certain embodiments where the resulting complex is to be administered to a mammal, the TNF-α can be a TNF-α characteristic of the species to which the complex is to be administered thereby reducing the likelihood of generating an immune response.

III. Combined uses.

[0089] The anti-HER2/neu-TNF-α compositions of this invention and the multivalent anti-HER2/ne« compositions of this invention are useful for enhancing wound healing and/or reducing or preventing scar and/or adhesion formation. The compositions can be used to prevent the formation of a scar at the same time as promoting wound healing. Alternatively, the compositions may be used for preventing scar formation by reducing or initiating regression of existing scars. Scar tissue as used herein refers to the fiber rich formations arising from the union of opposing surfaces of a wound. [0090] The compositions and methods of the invention may also include additional therapeutic and/or pharmacologically acceptable agents. For instance, the compositions or methods may involve other agents for the treatment of wounds such as, for instance, dexpanthenol, growth factors, enzymes or hormones, povidon-iodide, fatty acids, such as cetyl pyridinium chloride, antibiotics, analgesics, and the like.

[0091] Such factors include, but are not limited to, fibroblast growth factor (FGF),

FGF-I, FGF-2, FGF-4, FGF-10, VEGF, IL2, IL6, IL-10, platelet-derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-I, IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-α, TGF-β, HGH, cartilage inducing factors- A and -B, osteoid-inducing factors, osteogenin and other bone growth factors, collagen growth factors, heparin-binding growth factor-1 or -2, and/or their biologically active derivatives.

IV. Pharmaceutical Compositions.

[0092] In certain embodiments this invention provides pharmaceutical compositions comprising one or more of the chimeric moieties described herein. The compositions are typically formulated to deliver the chimeric moieties in effective amounts. An effective amount is that amount that alone or together with further doses or therapeutics produces the desired response, e.g., promoting wound healing, and/or reducing scar formation. In certain embodiments, this amount may involve a slowing the growth of a wounds or in the development of additional wounds. Preferably, however, it results in a reduction in wound size and/or an increase in the rate of wound healing. In certain embodiments this amount may involve a slowing in the progression of scar formation although more preferably, it may involve halting altogether the progression of scar formation

[0093] The actual amount delivered, of course, will depend upon the severity of the condition, the individual patient parameters, including age, physical condition, size, weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgement. It will be understood by those of ordinary skill in the art, however, that a patient may insist on a lower dose or tolerable dose for medical reasons, physiological reasons, or for virtually any other reasons.

[0094] The pharmaceutical compositions preferably are sterile for administration to a patient. When administered, the compositions are applied in pharmaceutically acceptable amounts and pharmaceutically acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but not pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically accept salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potasium, or calcium salts.

[0095] A large number of dressings, bandages, swabs, and topic medicaments are available for the treatment of wounds. These products fall into two categories, passive and active. Passive wound dressings are dressing which serve only to provide mechanical protection and a barrier to infection. The dressings themselves do not supply any composition which enables or facilitates the healing process of the wound. Examples of passive dressings include gauze and adhesive bandages. Active dressings are dressing which supply some biologically active compound to the site of a wound. One type of active dressing is a dressing or wrapping which delivers or has been impregnated with antimicrobials (e.g., Bacitracin). Another family of dressings which contain both passive and active properties are the hydrogels or hydrocolloids. Although many of these dressings do not supply any biologically active compound to the wound, they are specifically designed to create a moist environment around the wound to promote wound healing. Hydrogel and hydrocolloid dressings have been formulated to antimicrobials to help prevent and/or treat infection. However, to date, hydrogels or hydrocolloids have not been formulated with components that actively promote wound healing. In certain embodiments the chimeric moieties of this invention can be formulated for incorporation into such dressings, bandages, swabs, topical medicaments, ointments, and the like.

[0096] In various embodiments the chimeric moieties of this invention can be useful for parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The chimeric moieties can be formulated into pharmacological compositions {e.g., combination with an appropriate excipient). The pharmacological compositions can be administered in a variety of unit dosage forms depending upon the method of administration.

[0097] In certain embodiments, the chimeric moieties are provided as sprays, creams, salves, or ointments for topical use. For opthamalogical use they can be formulated in excipients for administration to the surface of an eye {e.g., eye drops). For surgical use, the compositions can be formulated for administration to a surgical site. In certain embodiments the composition can be incorporated into, e.g. biodegradable time-release matrices that are left implanted in a surgical site. [0098] In certain embodiments a typical pharmaceutical composition for administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used in certain instances. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., (1980) Mack Publishing Company, Easton, Pennsylvania.

V. Kits.

[0099] In certain embodiments, this invention provides for kits for the treatment of wounds including, but not limited to traumatic/acute and/or recurrent wounds of the epithelia, mucosa, and/or various internal organs and tissues. Kits typically comprise a container containing a chimeric moiety of the present invention {e.g., anti-HER2/rce«-TNF- α). The chimeric moiety can be present in a pharmacologically acceptable excipient. In certain embodiments the kit will comprise a dressing {e.g. a sterile dressing), and/or a surgical swab impregnated with a chimeric moiety of the present invention. In certain embodiments the container is a container for aerosol administration of the chimeric moiety. [0100] In addition the kits can optionally include instructional materials disclosing means of use of the chimeric moiety (e.g. to enhance wound healing, and/or to reduce scar tissue formation, and/to reduce adhesion formation, etc.). The instructional materials may also, optionally, teach preferred dosages, counterindications, and the like. [0101] The kits can also include additional components to facilitate the particular application for which the kit is designed. Thus, for example, the kits can additionally comprise means for disinfecting a wound, for reducing pain, for attachment of a dressing, and the like.

[0102] While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

EXAMPLES

[0103] The following examples are offered to illustrate, but not to limit the claimed invention.

Example 1

Anti-human HER2/»e«-TNFA: an agonistic antibody inducing HER2/neu activation in vitro

[0104] We have previously described the production of an anti-human HER2/neu and murine TNFα fusion protein (ScFv-anti-hHER2-TNFα) to be used to deliver TNFα to the tumor site. ScFv-anti-hHER2-TNFα forms the trimeric structure essential for the biological activity of TNFα and exhibits high affinity binding to the HER2/neu tumor antigen as a result of the presence of three Fv moieties. To investigate the in vitro cytotoxicity of ScFv-anti-hHER2-TNFα against HER2/neu expressing tumor cell lines, SKBR3, a human breast cancer cell line expressing high levels of HER2/neu was treated with ScFv-anti-hHER2-TNFα in the presence of actinomycin D. Unexpectedly, ScFv-anti- hHER2-TNFα treated SKBR3 cells survived better than cells treated with control medium, 006/013815

indicating that ScFv-anti-hHER2-TNFα can protect SKBR3 cells against the apoptotic effect of actinomycin D.

[0105] To determine which aspect of ScFv-anti-hHER2-TINEFα contributes to the anti-apoptotic effect, SKBR3 were treated with murine TNFα, TAVC, (an IgG3 antibody with the same variable region as ScFv-anti-hHER2-TNFα), and ScFv-anti-hHER2- mutantTNFα(identical to ScFv-anti~hHER2-TNFα except lacking TNFα activity). No significant protection was observed in SKBR3 cells treated with murine TNFα or TAVC. However, ScFv-anti-hHER2-mutantTNFα and ScFv-anti-hHER2-TNFα exhibited a similar anti-apoptotic effect, suggesting that the presence of three ScFv moieties is essential for the anti-apoptotic effect.

[0106] To determine if ScFv-anti-hHER2-TNFα and ScFv-anti-hHER2- mutantTNFα activates the HER2/neu signal pathway, phosphorylation of HER2/neu, AKT and MAPK was determined at different times following treatment of SKBR3 cells with different TNFα fusion proteins. ScFv-anti-hHER2-TNFα and ScFv-anti-hHER2- mutantTNFα elicited enhanced HER2/neu phosphorylation within 5 minutes. Furthermore, both antibodies induce more robust AKT and MAPK activation in SKBR3 cells compared to TAVC and TANL (a IgG3 antibody with the same variable region as Herceptin).

[0107] To determine if AKT or MAPK kinase activation contributed to the agonistic effect of ScFv-anti-hHER2-TNFα and ScFv-anti-hHER2-mutantTNFα, fusion proteins treated SKBR3 cells were incubated with either an AKT inhibitor, a MAPK inhibitor or both in the presence of actinimycin D. Both the AKT inhibitor and the MAPK inhibitor significantly decreased the agonistic effect of ScFv-anti-hHER2-TNFα and ScFv-anti- hHER2-mutantTNFα, and the agonistic effect completely abrogated when both inhibitors were added to the fusion proteins treated SKBR3 cells. Therefore, both the AKT and MAPK pathways contribute to the agonistic effect of the TNFα fusion proteins. In summary, we have found that an anti-human HER2/neu and murine TNFα fusion protein functions as an agonistic for HER2/neu over-expressing cells with this agonistic effect dependent on the trimeric ScFv moiety of the fusion protein rather than the biological activity of TNFα.

Example 2 A trimeric anti-HER2/raea: ScFv and TNF-α fusion protein induces ΗER2/neu signaling and facilitates repair of injured epithelia

[0108] A novel antibody fusion protein (anti-HER2/neu ScFv-TNF-α) with TNF-α genetically fused to the carboxy terminus of a single chain Fv (ScFv) antibody specific for the human ΗER2/neu forms a homotrimeric structure via the noncovalent interactions of the TNF-α moiety and retains both TNF-α activity and the ability to bind ΗER2/neu. Surprisingly anti-HER2/ne« ScFv-TNF-α was shown to elicit a potent agonistic effect in ΗER2/neu overexpressing SKBR3 cells. In contrast to anti- ΗER2/neu IgG3, anti- ΗER2/neu ScFv-TNF-α inhibited the apoptosis induced by Actinomycin D in ΗER2/neu overexpressing cells. The anti-apoptotic effect resulted from the activation of ΗER2/neu and the downstream MAPK and Akt pathways. Remarkably, anti-ΗER2/neu ScFv-TNF-α facilitated the repair of injured epithelia. This accelerated wound healing required binding to ΗERlfneu but did not require TNF-α activity since anti-HER2/7iew ScFv-TNF-α (S 147Y), an anti-HER2/neu ScFv containing a mutant TNF-α with significantly decreased biological activity, demonstrated an equivalent anti-apoptotic effect and ability to facilitate wound healing. These results strongly suggest that anύ-ΗER2/neu ScFv-TNF-α can be used in the clinic to facilitate wound healing. Furthermore, mutant human TNF-α may be broadly used to trimerize ScFv antibodies producing polymeric antibodies with high avidity for antigen.

Introduction. [0109] The use of a tumor specific antibody as a targeting vehicle to deliver higher doses of TNF-α to the tumor site is one approach for improving therapeutic index of TNF- α. Since the trimeric structure of TNF-α is essential for its biological activity, it is unlikely that TNF-α fused to either the amino or carboxy terminus of the immunoglobulin heavy chain would be active. However, a single chain Fv (ScFv) fusion with TNF-α maintained both antigen binding specificity and TNF-α activity (Cooke et αl. (2002) Bioconjug Chem 13:7-15).

[0110] In the present study, we constructed a fusion protein consisting of the anti-

ΗER2/neu ScFv, C6MH3-B1, (Schier et αl. (1996) J. MoI. Biol, 263: 551-567) and TNF-α (anti-HER2/new ScFv-TNF-α), and investigated its effect oήΗER2/neu overexpressing cells. Unexpectedly, the trimeric anti-HER2/new ScFv-TNF-α exhibited robust activation of ΗER2/neu as well as the downstream MAPK and Akt pathways in ΗER2/neu overexpressing cells. In addition, it protects HER2/new overexpressing cells against Actinomycin D induced apoptosis, and remarkably, facilitates the repair of injured epithelia. These results strongly indicate that anti~HER2/neu ScFv-TNF-α may be useful for facilitating wound healing.

Materials and methods.

Cell lines and culture conditions.

[0111] The Chinese hamster ovary (CHO) cell line Pro-5 (American Type Culture

Collection [ATCC], Manassas, VA) and its derivatives expressing ScFV-TNF-α fusion proteins were cultured in IMDM (Irvine Scientific, Irvine,CA) supplemented with 2 mM L- glutamine, 10 U/ml penicillin, lOμg/ml streptomycin (GPS) (Sigma Chemical, St. Louis, MO) and 5% calf serum (Atlanta Biologicals, Norcross, GA). Murine myeloma cell lines Sp2/0 (ATCC), P3X63Ag8.653 (ATCC) and their derivatives expressing anti-HER2/72ew IgG3 and D2F2/E2, a murine mammary cell line expressing human ΗER2/neu on the cell surface were grown in IMDM supplemented with 10% calf serum and GPS. J-774 A.I, a murine macrophage cell line (ATCC), human breast cancer cell line SKBR3 (ATCC) and L929 fibroblast (ATCC) were cultured in IMDM with 5% calf serum and GPS. The human colonic epithelial cell line Caco2 (ATCC) was maintained in high-glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% calf serum and GPS.

Plasmid construction.

[0112] The construction and characterization of anti-HER2/πβw IgG3 composed of the heavy and light chain variable regions of the humanized Ab 4D5-8 (rhuMab HER2, Herceptin, Genentech, San Francisco, CA) and the constant region of human IgG3 has been previously described (DeIa Cruz et αl. (2000) J. Immunol, 165:5112-5121). To clone murine TNF-α, J-774 A.I was stimulated 4 hr with 5 μg/ml LPS (Sigma) in IMDM. Total mRNA was purified and murine TNF-α including its leader sequence was amplified by PCR using the following primers: 5'-GGG ATA TCC ACC ATG AGC ACA GAA AGC ATG- 3' (SEQ ID NO:1) and 5'-CCT GAT CAC AGA GCA ATG ACT CCA AAG-3' (SEQ ID NO:2). The PCR product was cloned into the TA Cloning Vector (Invitrogen, Carlsbad, CA). To construct murine TNF-α lacking a leader sequence, the 5' primer with an EcoRl site ( 5'-CGG AAT TCG CTC AGA TCA TCT TCT CAA AAT TC-3\ SEQ ID NO:3) and the same 3' primer described above were used for PCR. The PCR product was Ii gated into the TA Cloning Vector, sequenced, and pTA-TNFα with the correct TNF-α sequence was digested with EcoRl and BamHl to release the DNA fragment containing the mature sequence of murine TNF-α which was inserted into pASK-EB A4 Strep-tag II (Sigma). pucC6MH3-B 1 expressing an anti-human HER2/rceu ScFv with high binding affinity was used. The heavy and light chain variable regions of C6MH3-B1 ScFv were amplified by PCR and using the appropriate restriction sites, inserted into the human γ3 heavy chain (pAH4802) and K light chain (pAG4622) expression vectors respectively (Coloma et al. (1992) J Immunol Methods 152:89-104). To construct the anti-human ΗER2/neu ScFv (C6MH3-B1)- TNF-α fusion protein, overlap PCR was first used to introduce a sequence encoding the NWSHPQFEK streptavidin tag (SEQ ID NO:4) and GGGGSGGGGSGGGGS peptide linker (SEQ ID NO: 5) upstream of the mature murine TNF-α gene with the forward primer 5'-GTC ACA TCC GCA GTT CGA GAA ATC AGG TGG TGG CGG TTC AGG CGG AGG TGG CTC TGG CGG TGG CGG ATC GCT CAG ATC ATC TTC TCA AAA TTC-3' (SEQ ID NO:6) and the reverse primer 5'-CCT GAT CAC AGA GCA ATG ACT CCA AAG-3' (SEQ ID NO:7). The product was used as template for a second PCR using the same reverse primer and a second forward primer 5'-GTC CTA GGT CGT AAC TGG TCA CAT CCG CAG TTC GAG AAA-3 ' (SEQ ID NO:8). The final PCR product was ligated into the TA vector. The vector, after sequencing, was digested with EcoRV and Avrll to release the DNA fragment containing murine TNF-α gene which was inserted into a TA vector containing C6MH3-B1 3' of the ScFv gene. The resulting plasmid was digested with EcoRV and BamHl and the fragment containing C6MH3-B1 ScFv joined to murine TNF-α was inserted into a pcDNA3.1 vector (Invitrogen), in which the neomycin resistance gene was replaced with a histidinol resistance gene. The resulting plasmid, designated p9606, contains the coding region for C6MH3-B1 ScFv followed by a NWSHPQFEK streptavidin tag (SEQ ID NO:4), the GGGGSGGGGSGGGGS peptide linker (SEQ ID NO:5) and mature murine TNF-α. To create a mutant murine TNF-α protein, the forward primer 5'-CTG CCC GTA CTC CGC AAA G-3' (SEQ ID NO:9) and reverse primer 5'-GGA GTG GCT GAG CCA GCG C-3' (SEQ ID NO:10) were used to introduce a point mutation at TNF-α residue 147(Ser→Tyr). The resulting plasmid was digested with Avrll and BamHl and the DNA fragment containing murine TNF-α(S147Y) inserted at the end of C6MH3-B1 ScFv. The resulting plasmid designated as p9609 contains the coding region for C6MH3-B1 ScFv followed by a NWSHPQFEK streptavidin tag (SEQ ID NO:4), GGGGSGGGGSGGGGS peptide linker (SEQ ID NO:5) and murine TNF- α(S 147Y). The construction and characterization of an anti-dansyl ScFv antibody has been previously described (Coloma and Morrison (1997) Nat. Biotechnol, 15: 159-163). To construct anti-dansyl ScFv-TNF-α, the forward primer 5'-CTA GCT AGC GGT GGC GGT GGC TCG GGC GGA GGT GGG TCG GGT GGC GGC GGA TCT GAT GTT -3' (SEQ ID NO: 11) and the reverse primer 5'-CTC GAA CTG CGG ATG TGA CCA GTT AAC ACG TTT TAT TTC CAA CTT TGT CC -3' (SEQ ID NO: 12) were used to generate a fragment encoding the light chain variable region of anti-dansyl ScFv and the NWSHPQFEK streptavidin tag (SEQ ID NO:4); the forward primer 5'-GTT AAC TGG TCA CAT CCG CAG TTC GAG AAA-3' (SEQ ID NO: 13) and the reverse primer 5'-CGG GAT CCT CAC AGA GCA ATG ACT CCA AAG-3' (SEQ ID NO: 14) were used to generate the second DNA fragment containing the NWSHPQFEK streptavidin tag (SEQ ID NO:4), GGGGSGGGGSGGGGS peptide linker (SEQ ID NO:5) and the mature sequence of murine TNF-α. Using the two DNA fragments as templates, a PCR with the forward primer 5'-CTA GCT AGC GGT GGC GGT GGC TCG GGC GGA GGT GGG TCG GGT GGC GGC GGA TCT GAT GTT-3' (SEQ ID NO: 15) and the reverse primer 5'-CGG GAT CCT CAC AGA GCA ATG ACT CCA AAG-3' (SEQ ID NO: 16) was used to generate a DNA fragment containing the light chain variable region of anti-dansyl ScFv followed by a NWSHPQFEK streptavidin tag (SEQ ID NO:4), GGGGSGGGGSGGGGS peptide linker (SEQ ID NO:5) and murine TNF-α. The resulting DNA fragment was ligated into the TA vector and after sequencing was digested with Nhel and BamHl to release the DNA fragment containing the light chain variable region of anti-dansyl ScFv followed by a

NWSHPQFEK streptavidin tag (SEQ ID NO:4), GGGGSGGGGSGGGGS peptide linker (SEQ ID NO: 5) and murine TNF-α gene. This fragment was inserted at the end of heavy chain variable region of anti-dansyl ScFv. It generated a pcDNA3.1 vector containing anti- dansyl ScFv followed by a NWSHPQFEK streptavidin tag (SEQ ID NO:4), GGGGSGGGGSGGGGS peptide linker (SEQ ID NO:5) and murine TNF-α. Production and purification of different recombinant proteins.

[0113] p9606, p9609 and p9622 vectors were transfected in the CHO cell line Pro-5 using the lipofectamine plus reagent (Invitrogen). Stable transfectants were selected with 1 mM histidinol (Sigma) and the highest producers were identified using a ELISA plate coated with rat anti-mouse TNF-α (BD Biosciences, San Jose, CA) and detected by biotinylated rat anti-mouse TNF-α (BD Biosciences). Transfectants were expanded in 150 x 25 mm tissue culture dishes (BD Biosciences) containing protein free CHO liquid soy medium (HyClone, Logan, UT), and the culture supernatants were concentrated with an Amicon stirred ultrafiltration cell (Amicon, Beverly, MA). Transfectants producing anti- ΗERl/neu (C6MH3-Bl)-IgG3 were selected and characterized as previously described (DeIa Cruz et al. (2000) J. Immunol, 165: 5112-5121). The anti-HER2/ne« (C6MH3-B1) IgG3 antibody was purified from culture supernatants using protein G immobilized on Sepharose 4B fast flow (Sigma). Purity and integrity were assessed by Coomassie blue staining of proteins separated by SDS-PAGE. The production and purification of soluble ΗER2/neu has been previously described (DeIa Cruz et al. (2003) Vaccine 21 : 1317-1326).

Protein cross-linking.

[0114] 9 μl of PBS was mixed with 10 μl of 10 μM TNF-α fusion protein and 1 μl of a freshly made solution of 6.35 mM EGS [ethylene glycolbis (succinimidylsuccinate)] (Pierce, Rockford, IL). After 30 minutes incubation at room temperature, 1 μl of 1 M glycine was added and the solution was incubated for another 30 minutes. 5 μl of 5 X SDS sample buffer and 1 μl of 2-mercaptoethanol (Fisher Scientific, Hampton, NH) were then added, samples boiled at 95°C for 5 minutes, and 25 μl aliquots fractionated on a 12% SDS- PAGE. Proteins were visualized by western blot using biotinylated rat anti-mouse TNF-α.

FPLC. [0115] Purified TNF-α fusion proteins and standard proteins were analyzed in a 0.5

M NaCl / 20 mM phosphate solution, pH 6.5, using a Superose 6HR 10/30 column (Amersham Pharmacia Biotech, Piscataway, NJ.) at a flow rate of 0.25ml/min. The injection volume of 100 μl contained 40 μg of protein. Flowcytometry analysis.

[0116] To detect the reactivity of various ScFv-TNF-α fusion proteins with

D2F2/E2 cells, 1 x 10⁶ cells were incubated at 4°C for 1 hour with 10 pM of the fusion protein. Cells were then reacted with biotinylated rat anti-mouse TNF-α (BD Biosciences) diluted 1:35. The bound biotinylated Abs were detected with PE-labeled streptavidin (BD Biosciences) diluted 1:1500 and analyzed by flow cytometry using a FACScan (Becton Dickinson).

TNF-α cytotoxicity activity.

[0117] L-929 cells were plated in a 96-well tissue culture plate (Falcon, Lincoln Park,NJ) at a density of 4 x 10⁴ cells/well and incubated overnight at 37°C in a 5% CO₂ atmosphere. Afterward, serial dilutions of different ScFv-TNF-α fusion proteins or recombinant murine TNF-α were added in the presence of Actinomycin D (8 μg/ml, A.G. Scientific, San Diego, CA), and the plate incubated for 24 hours. Surviving adherent cells were then stained with 50 μl of crystal violet (0.05% in 20% ethanol) for 10 min. The plates were washed with water and the remaining dye solubilized by the addition of 100 μl of 100% methanol. Plates were read on an ELISA reader at 595 nm.

MTS assay for the anti-apoptotic effect of anti-HER2/neu ScFv-TNF-α.

[0118] SKBR3 cells were plated in a 96-well tissue culture plate at a density of 4 x

10⁴ cells/well and incubated overnight at 37°C in a 5% CO₂ atmosphere. Afterward, serial dilutions of different ScFv alone or with the indicated competitive antibodies were added in the presence of Actinomycin D (4 μg/ml) and the plate incubated overnight. For some experiments, the fusion protein treated SKBR3 cells were incubated with U0126 (Calbiochem, San Diego, CA) and/or LY294002 (Calbiochem) in the presence of Actinomycin D (4 μg/ml) overnight. Plates were then developed by addition of 20 μl of MTS solution (Promega, Madison, WI) and measured on an ELISA reader at 490 nm.

Western blot analysis.

[0119] SKBR3 cells were treated with different fusion proteins or antibodies for the indicated times, washed with ice-cold PBS, and lysed on ice for 10 min in lysis buffer (0.125% Nonidet P-40, 0.875 % Brij 97, 10 mM Tris-HCl, pH7.5, 2 mM EDTA, 0.15 M NaCl, 0.4 mM Na₃Vo₄, 0.4 mM NaF, 1 mM PMSF, 2.5 μM leupeptin, 2.5 μM aprotinin). Cell lysates were clarified at 10,000 x g for 10 min at 4°C. Protein samples were then boiled in sample buffer before separation on 8% SDS-PAGE gels and transfer onto PVDF microporous membranes (Millipore, Billerica, MA). After blocking with 3% bovine serum albumin in 150 mM NaCl, 50 mM Tris-HCl, pH 7.6 (TBS) for 1 hour at room temperature, blots were probed with the indicated primary antibodies overnight at 4°C. The blots were then washed 3 times at room temperature with 0.05% Tween 20 in TBS, incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP), and detected by a peroxidase-catalyzed enhanced chemiluminescence detection system (ECL; Pierce). In order to confirm equal loading of proteins, blots that had been probed for the phosphorylated proteins were stripped and reprobedwith an antibody against an appropriate control protein. For this procedure, 10 ml of stripping buffer, consisting of 2% (w/v) SDS, 62.5 mM Tris, pH 6.7 and 100 mM 2-mercaptoethanol, was added to the membrane for 15 min with constant shaking at 60⁰C. The membrane was then washed (6 x 5 minutes in TBS), blocked and probed with the appropriate primary antibody.

Antibodies for western blot analysis

[0120] Monoclonal anti-phosphotyrosine 4G10 was obtained from Upstate

Biotechnology Inc. (UBI, Lake Placid, NY). Anti-HER2/new antibody sc-284, a rabbit polyclonal antibody against the carboxy terminus of human ΗERl/neu, was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Anti-Akt, anti-phosphoAkt (Ser473), anti- p44/42 MAPK and anti-phosphop44/42 MAPK (ElO) were obtained from Cell Signaling Technology Inc. (Beverly, MA). Polyclonal HRP-conjugated rabbit anti-mouse IgG was obtained from ICN Pharmaceuticals Inc. (Aurora, Ohio). Polyclonal HRP-conjugated donkey anti-rabbit IgG was obtained from Amersham Biosciences (Piscataway, NJ).

Wounding and measurement of wound repair on polarized epithelia

[0121] Caco2 cells were plated in a 24-well tissue culture plate (Falcon) at a density of 2.5xlO⁵ cells/well and incubated 72 hour at 37°C in a 5% CO₂ atmosphere for polarization. The bottom of a yellow tip (Fisher Scientific) was used to generate a consistent injury area on the polarized cell layer. The injured epithelium was treated with 100 nM of fusion protein or antibodies for the indicated times, and the wound photographed at different times using a Nikon Phase Contrast inverted microscope and a 3CCD camera (Toshiba, New York, NY). The width of each wound was measured at 3 sites in each image, and the percentage of wound recovery was calculated by comparison with the wound width at time 0. Statistical analysis was made using a two-tailed Student's t test. Results were regarded as significant if P values were < 0.05.

Results

Production and characteriztion of anti-HER2/neu ScFv- TNF-α and anti-

HER2/neu ScFv-TNF-α(S147Y) [0122] TNF-α forms a trimeric structure, which is important for its biological activity. We explored a strategy in which we fused an anti-HER2/n<?« ScFv (C6MH3-B1, that binds ΗERllneu with high affinity (Schier et at. (1996) JMoI Biol 263:551-567)) to the amino terminus of mature murine TNF-α, using a flexible [(GIy₄) Ser]₃ (SEQ ID NO:5) linker and a NWSHPQFEK streptavidin tag (SEQ ID NO.4) to separate the two protein moieties. A schematic representation of the proposed trimeric anti-HER2/new ScFv-TNF-α is shown in Figure 1. To construct a control antibody with the same antigen binding specificity but lacking TNF-α activity, we constructed an anti-HER2/new ScFv fusion containing TNF-α with a point mutation at residue 147 (Ser→Tyr). TNF-α(S147Y) has been shown to exhibit a 100 fold decrease in TNF-α biological activity while still maintaining a trimeric structure (Zhang et at. (1992) J. Biol. Chem. 267:24069-24075). In addition, we also expressed an anti-dansyl ScFv-TNF-α fusion protein and recombinant murine TNF-α (rTNF-α).

[0123] The purified proteins were analyzed by SDS-PAGE under reducing conditions (Figure 2A). rTNF-α migrated with an apparent molecular weight of 17 KDa, and all three ScFv-TNF-α fusion proteins migrated at an apparent molecular weight of 47 KDa, the expected size for the monomeric fusion protein. The double bands of the ScFv- TNF-α fusion proteins were due to variability in the N-linked glycosylation of murine TNF- α since the proteins secreted by tunicamycin treated transfectants showed only a single band on SDS-PAGE (data not shown). [0124] To determine whether the ScFv-TNF-α fusion proteins form trimers, anti-

ΗER2Ineu ScFv-TNF-α and anti-HER2/rceu ScFv-TNF-α(S147Y) were treated with the crosslinking agent EGS [ethylene glycolbis (succinimidylsuccinate)], separated by SDS- PAGE and visualized by western blot analysis. Dimer and trimer forms of anti-HER2/?ieκ ScFv-TNF-α and anti-HER2/n<?« ScFv-TNF-α(S 147Y) were observed (Figure 2B); similar results are seen following crosslinking of wild type TNF-α (Van Ostade et al. (1991) Embo J 10:827-836). FPLC analysis confirmed the trimeric structure of the anti-HER2/rcew ScFv- TNF-α and anti-HER2/rceu ScFv-TNF-α(S147Y) as both eluted in the fractions expected for the 141 KDa trimers. (Figure 2C).

Antigen binding and cytotoxic activity of anti-HER2/neu ScFv-TNF-α.

[0125] To investigate the functional activity of anti-HER2/new ScFv- TNF-α, we first tested its binding to a murine mammary cell line, D2F2/E2, which expresses high levels of human ΗER2/neu. Both anti-HER2/new ScFv- TNF-α (Figure 3A, panel 1) and anti-HER2/rceu ScFv-TNF-α(S 147 Y) (Figure 3 A, panel 2) bound D2F2/E2 cells while anti- dansyl ScFv- TNF-α did not, excluding the possibility that binding was through the TNF-α receptor (Figure 3 A, panel 3). This result demonstrated that the ScFv moiety of anti- BER2/neu ScFv-TNF-α and anti-HER2/7ϊe« ScFv- TNF-α(S147Y) retains its ability to bind human ΗER2/neu.

[0126] The cytotoxicity of rTNF-α and ScFv- TNF-α was assessed using murine L- 929 cells. As shown in Figure 3B, rTNF-α, anti-HER2/new ScFv-TNF-α and anti-dansyl ScFv- TNF-α exhibited similar cytotoxicity against L-929. As predicted, anti-HER2/7ieM ScFv- TNF-α(S147Y) exhibited decreased cytotoxicity against L929 cells. The IC₅₀ values were 2.5, 4 and 6 pM for rTNF-α, anti-dansyl ScFv- TNF-α and anti-HER2/neu ScFv-TNF- α respectively, and 300 pM for anti-HER2/«gw ScFv-TNF-α(S147Y). Therefore, TNF-α retained its biological activity when fused to the scFvs although there was a slight reduction in specific activity.

Anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S147Y) induce a potent anti-apoptotic effect in SKBR3 cells via HER2/neu binding.

[0127] Although TNF-α has been shown to elicit a direct cytotoxic effect in some tumors (Laster et al. (1988) J. Immunol, 141: 2629-2634), it has been demonstrated that HER2/neu activation can block the apoptosis induced by TNF-α by activating the Akt/NF- KB pathway in ΕERllneu overexpressing tumors including SKBR3 (Zhou et al. (2000) J. Biol. Chem., 275: 8027-8031).

[0128] Treatment with Actinomycin D was cytotoxic to SKBR3 cells with O.D ₄go values of 0.85 and 2.0 following MTS addition for the Actinomycin D treated cells and control medium treated cells, respectively (data not shown). Unexpectedly, in the presence of Actinomycin D, anti-HER2/new ScFv-TNF-α treated SKBR3 cells survived better than cells treated with medium alone (Figure 4A). Anti-HER2/rcew ScFv-TNF-α(S147Y) exhibited similar activity indicating that it was ΗER2/neu binding and not the biological activity of TNF-α that was responsible for this protective effect. Consistent with this conclusion, no protection against the apoptotic effect of Actinomycin D was seen when SKBR3 cells were treated with rTNF-α (Figure 4A). The protection against Actinomycin D induced apoptosis is only seen with the trimeric ScFv moiety, since an IgG3 antibody with the same variable region as the anti-HER2/new ScFv-TNF-α, did not exhibit this effect (Figure 4A). Additionally, increasing concentrations of anti-HER2/neu IgG3 but not anti- dansyl IgG3 abolished the anti-apoptotic effect against Actinomycin D induced by anti- HER2/rceu ScFv-TNF-α and anti-HER2/n<?w ScFv-TNF-α(S 147 Y) (Figure 4B). These results suggest that trimeric anti-HER2/new ScFv initiates a cellular resistance mechanism against the apoptotic effect of Actinomycin D by cross-linking ^~HER2/neu on SKBR3 cells.

Anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S147Y) induced tyrosine phosphorylation of HER2/neu.

[0129] To investigate potential mechanisms for the resistance to the apoptotic effect of Actinomycin D induced by the trimeric anti-HER2/neu ScFv in ΗER2/neu expressing cells, the phosphotyrosine content of ΗER2/neu was examined in SKBR3 cells treated with these antibodies. In the presence of control medium, a protein with the molecule size of 185 kD but not any other proteins with the molecule size between 97 kD and 220 kD was phosphorylated on the tyrosine residue , which is expected to be the activated ΗER2/neu (Figure 5). In addition, both anti-HER2/W ScFv-TNF-α (Figure 5A) and anti-HER2/neu ScFv-TNF-α(S 147Y) (Figure 5B) at concentrations of 50 nM and 100 nM induced a strong increase in ΗERllneύ¹ s phosphotyrosine content by 5 min while the extent of tyrosine phosphorylation in the presence of 100 nM rTNF-α was the same as that seen in the presence of control medium. This result suggests that activation of ΗERllneu initiated by the trimeric anti-HER2/7ieu ScFv antibodies may contribute to resistance against the apoptotic effect of Actinomycin D.

Anti-HER2/neu ScFv-TNF-α induced robust activation of p44/42 MAPK (ERK1+2) and Akt.

[0130] The mitogen-activated protein kinase (MAPK) and PBK pathways are the major signaling cascades downstream of activated ErbB receptors including ΕEKllneu (Olayioye et al. (2000) EMBO J., 19: 3159-3167). Activation of these pathways has been shown to result in cellular proliferation and resistance to apoptosis in ΗER2/neu expressing tumor cells (Zhou et al. (2000) /. Biol. Chem., 275: 8027-8031; Leung et al. (2004) MoI. Cancer 3:15). As shown in Figure 6, both anti-HER2/7ϊeu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S 147Y) initiated robust ERK1+2 phosphorylation in SKBR3 cells by 2 min. ERK1+2 phosphorylation showed an 8 fold increase which persisted for at least 10 min in SKBR3 cells treated with either anti-HER2/rcew ScFv-TNF-α or anti-HER2/new ScFv-TNF- α(S 147 Y). In contrast, IgG3 antibodies with the variable regions of C6MH3-B 1 or

Herceptin, induced weak and transient ERK1+2 phosphorylation in SKBR3 cells (Figure 6). Anti-HER2/new ScFv-TNF-α and anti-HER2/new ScFv-TNF-Ct(S 147Y) also induced significant phosphorylation of Akt within 30 sec (Figure 7). By 10 min the Akt phosphorylation was 2.7 and 2.2 fold increased in SKBR3 cells treated with anti-HER2//ιeM ScFv-TNF-α and anti-HER2/new ScFv-TNF-α(S 147 Y), respectively. Akt phosphorylation persisted in anti-HER2/rcew ScFv-TNF-α treated SKBR3 cells for over 60 min, while the increased Akt phosphorylation in anti-HER2/new ScFv-TNF-α(S147Y) treated SKBR3 cells was barely detectable by 30 min. There was very little Akt activation in the anti-HER2/røeu IgG3 treated SKBR3 cells (Figure 7). These results demonstrate that the trimeric anti- ΗER2/neu ScFv antibodies can initiate potent ERK1+2 and Akt activation downstream of activated ΗER2/neu in SKBR3 cells. Activation of both MAPK and Akt contribute to the anti-apoptotic effect induced by anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF- α(S147Y).

[0131] To further investigate whether ERK1+2 and/or Akt activation induced by the trimeric anti-HER2/neu ScFv antibodies contributed to the resistance of SKBR3 cells to Actinomycin D induced death, Actinomycin D treated SKBR3 cells were incubated with anti-HER2/neκ ScFv-TNF-α or anti-HER2/πew ScFv-TNF-α(S147Y) in the presence of the ERK1+2 inhibitor, U0126, and/or the Akt inhibitor, LY294002 for 24h (Figure 8). As seen previously, anti-HER2/neu ScFv-TNF-α or anti-HER2/nβκ ScFv-TNF-α(S147Y) significantly protected SKBR3 cells against the apoptosis induced by Actinomycin D (lanes 1-3). In the presence of LY294002, the protective effect was reduced (lanes 4-6). The protective effect exhibited by anti-HER2/7zeu ScFv-TNF-α or anti-HER2/neu ScFv-TNF- α(S 147Y) was also reduced by treatment with UO 126 (lanes 7-9). Importantly, the protection against Actinomycin D induced apoptosis mediated by anti-HER2/7ϊew ScFv- TNF-α or anti-HER2/nβ« ScFv-TNF-α(S 147 Y) was completely abolished when both

LY294002 (40 μM) and U0126 (10 μM) were present (lanes 10-12). Therefore, activation of ERK1+2 and Akt contributes to the anti-apoptotic effect induced by the trimeric anti- BER2/neu ScFv antibodies in SKBR3 cells.

Anti-HER2/neu ScFv-TNF-α and anti-HER2/neu ScFv-TNF-α(S147Y) facilitate the repair of mechanically injured epithelia

[0132] HER2/neu activation has been shown to participate in the repair of injured epithelia (Vermeer et at. (2003) Nature 422:322-326; Xu et at. (2004) Invest Ophthalmol Vis Sci 45:4277-4283). Trimeric mύ-ΗER2/neu ScFv also facilitates the repair of mechanical wounding in cultured human colonic epithelial cells by binding ΗERllneu (Figure 9). The wound in anti-HER2/neu ScFv-TNF-α or anti-HER2/rceu ScFv-TNF- α(S147Y) treated cells was almost completely healed by 29 h while the wound of the control medium treated cells was still apparent. Analysis of 6 independent experiments confirmed this result with a statistically significant (P< 0.001) increase in the speed of wound healing seen in anti-HER2/7ieu ScFv-TNF-α and anti-HER2/rceu ScFv-TNF- α(S147Y) treated cells (Table 1). In contrast, IgG3 antibodies with the same variable region as C6MH3-B1 or with the same variable region as Herceptin did not show any significant affect on the rate of wound healing compared to treatment with the PBS (Figure 9, Table 1). The enhancement of wound healing required the binding of the trimeric fusion proteins to ΗER2/neu on the injured epithelial cells, as it was inhibited by the presence of soluble ΗERllneu. The wound in anti-HER2/neu ScFv-TNF-α or anti-HER2/rcew ScFv-TNF- α(S147Y) treated cells (Figure 10, panels A and C) was almost healed within 25 h; in contrast, the rate of wound healing was significantly decreased when an excess of soluble BER2/neu was present (Figure 10, panels B and D). These results suggest that the trimeric anti-HER2/new ScFv antibodies but not bivalent IgG3 antibodies facilitate wound repair through the binding of ΗER2/neu on the injured epithelial cells.

[0133] Table 1. Anti-HER2/new antibodies and repair of wound mechanical wounding.

[0134] Injured epithelial cells were treated with 100 nM of anύ-ΗER2/neu antibodies for the indicated times. The injured epithelial monolayers were photographed using a phase contrast inverted microscope with a 3 CCD camera. The width of each wound was measured at 3 sites in each image, and the percentage of wound recovery was calculated by comparison with the original wound width. The percentage shown in the column was the mean of wound recovery from six independent images for each treatment at each time point. The numbers in the parentheses correspond to the SD of the measurement. The asterisks indicate P < 0.001 compared to the control of CHO medium or PBS.

Discussion

[0135] We have constructed and characterized a novel fusion protein with TNF-α fused to an anti-HER2/new ScFv containing the variable region of C6MH3-B1 (Schier et αl. (1996) J. MoZ. Biol, 263: 551-567). Surprisingly this fusion protein, anti-HER2/πew ScFv- TNF-α, exhibited a potent agonistic effect, activating ΗER2/neu and the downstream MAPK and PI3K signaling cascades. Remarkably this fusion protein also facilitated the repair of injured epithelial cell monolayers. These unique effects did not require the biological activity of TNF-α since anti-HER2/rcew ScFv fused to TNF-α (S 147Y) exhibited similar activities. The trimeric structure of anti-ΗER2/neu ScFv appears essential for initiating ΗER2/neu signaling and facilitating the repair of injured epithelia, since an IgG3 antibody with the same variable region as C6MH3-B1 did not show similar effects.

[0136] Anti- ΗER2/neu ScFv- TNF-α described in the present study appears to differ in its functional properties from TNF-α fused with a different anti-HER2/rceu variable region (sFv23/TNF) (Rosenblum et al. (2000) Int J Cancer 88:267-273). sFv23/TNF exhibited modest cytotoxicity against SKBR3 cells in the absence of Actinomycin D while anti- ΗER2/neu ScFv- TNF-α did not exhibit any significant effect on SKBR3 cells in the absence of Actinomycin D. Since antibodies recognizing different epitopes on the same antigen may exhibit different effects, activation of ΗER2/neu signaling and the facilitation of wound repair induced by anti-HER2/ne« ScFv-TNF-α and anti-HER2/rcew ScFv-TNF- α(S147Y) may require both their unique anti-HER2/neκ variable region, C6MH3-B1 and a trimeric structure.

[0137] The ERK signaling pathway, also known as the p44/42 MAP kinase pathway, is a major determinant in the control of cell growth and migration, and aberrantly active ERK signaling has been identified in many types of human tumors (PoIa et al. (2003) /. Biol. Chem. 278:40601-40606; Hoshino et al. (1999) Oncogene 18:813-822). ERK activation is essential for cell survival following oxidant injury (Guyton et al. (1996) /. Biol. Chem. 271:4138-4142) and NTH3T3 cells expressing constitutively active MEK (the immediate upstream regulator of ERK) were more resistant to oxidant toxicity (Id.). In addition, the migratory and invasive activity of human neuroblastoma cells was inhibited by using a ERK inhibitor, PD98059 (PoIa et al (2003) J. Biol. Chem. 278:40601-40606). In the present study, the trimeric anti-HER2/neM ScFv antibodies were found to induce robust and persistent ERK activation in HER2/neu expressing cells even when the activity of the attached TNF-α was greatly compromised. ERK activation was initiated within 30 sec and, remarkably, was maintained for at least 90 min (Figure 6). Therefore, proliferation and migration induced by ERK activation undoubtedly makes a major contribution to the enhancement of wound repair induced by the trimeric anti-HER2/new ScFv antibodies.

[0138] Activated ERK and Akt both contribute to the anti-apoptotic effect induced by the trimeric anti-HER2/neu ScFv antibodies. The presence of inhibitors of either ERK or Akt decreased the anti-apoptotic effect of the trimeric fusion proteins but this effect was completely abolished only in the presence of both ERK and Akt inhibitors (Figure 8). Although TNF-α has been shown to induce the phosphorylation of Akt in a variety of cells (Osawa et α/. (200I) J. Immunol, 167:173-180; Sandra et al. (2002) Cell Signal 14:771- 778), we did not observe significant enhancement of Akt activation in SKB R3 cells treated with the anti-dansyl ScFv-TNF-α fusion protein (data not shown). Nervertheless, anti- ΗERllneu ScFv-TNF-α but not anti-ΗER2/neu ScFv-TNF-α(S147Y) induced prolonged phosphorylation of Akt . Since activation of ΗERllneu has been shown to induce activation of the Akt/NF-κB pathway (Zhou et al. (2000) J. Bio.l Chem., 275:8027-8031), the ΗERllneu signaling induced by trimeric anti-HER2/new ScFv may sensitize SKBR3 cells to TNF-α stimulation, thus resulting in the prolonged phosphorylation of Akt observed. While &nti-ΗER2/neu ScFv-TNF-α induced prolonged activation of Akt compared with anti- HER2/new ScFv-TNF-α(S147Y), both anύ-HER2/neu ScFv-TNF-oc and anύ-ΗER2/neu ScFv-TNF-α(S147Y) exhibited a similar anti-apoptotic effect against Actinomycin D and facilitated the repair of injured epithelia with the same potency, indicating that the prolonged activation of Akt did not contribute to these effects.

[0139] Activation of the PI3K pathway is induced by the trimeric anti-HER2/ne«

ScFv antibodies as shown by the induction of phosphorylated Akt (Figure 7). Rac, a member of the Rho GTPases, has been shown to stimulate the migration of different type of cells (PoIa et αl. (2003) J. Biol. Chem. 278:40601-40606; Weiss-Haljiti et αl. (2004) J. Biol. Chem. 279:43273-43284), with PI3K activity necessary and sufficient for Rac activation (Hawkins et αl. (1995) CurrBiol 5:393-403). Therefore, it is likely that Rac induced cell migration contributed to the enhancement of wound repair.

[0140] Genetic fusion of antibody with a protein capable of forming noncovalent oligomers, for example avidin, has been shown to be an effective method for producing a polymeric antibody (Ng et αl. (2002) Proc Nαtl Acαd Sci USA 99:10706-10711). However, the immunogenicity of foreign proteins hampers the clinical use of these antibodies in humans. Use of a TNF-α with a point mutation at residue 87 (Tyr— >Ser) and lacking biological activity but maintaining its trimeric struture (Zhang et al. (1992) J. Biol. Chem. 267:24069-24075) provides a novel strategy to construct a polymeric antibody with minimal immunogenicity. [0141] Herceptin, a human IgGl antibody binding to the extracellular domain of

ΗER2/neu, has been shown to transiently induce a modest activation of HER2/neu signaling in vitro (Scott (1991) /. Biol. Chem., 266:14300-14305). In the present study, we have shown that both Herceptin and the human IgG3 antibody with the C6MH3-B1 variable region induced a similar transient and weak activation of ERK and Akt; however, the trimeric anti-HER2Mew ScFv antibodies with C6MH3-B1 variable region initiated a potent and persistent ΗER2/neu signaling and exhibited an anti-apoptotic effect in SKB R3 cells. Therefore, it is likely that cross-linking of multiple BER2/neu receptors is one of the critical determinants for these effects.

[0142] Without being bound to a particular theory, it is believed that a tetravalent anti-HER2/«ew ScFv recognizing the same epitope as the trimeric anti-HER2/πeu ScFv would be expected to activate more robust ΗER2/neu signaling by cross-linking more ΗER2/neu receptors.

[0143] The original goal of the present study was to use a tumor specific antibody to deliver high doses of TNF-α to ΗER2/neu expressing tumor cells. TNF-α exhibits a wide spectrum of biological activities including promoting cytolysis of some tumor cell lines (Laster et al. (1988) /. Immunol, 141:2629-2634), enhancing the anti-tumor effect of dendritic cells (Candido et al. (2001) Cancer Res., 61:228-236) and activating host immunity (Hocket al. (1993) Proc. Natl. Acad. ScL, USA, 90:2774-2778). Therefore, TNF- α fused antibodies could be a promising anti-cancer therapeutic. Although the TNF-α fused anti-HER2/τϊew ScFv we describe in this study activated robust ΗER2/neu signaling and protected SKBR3 cells against Actinomycin D induced apoptosis, it did not enhance the proliferation of SKBR3 cells during a long-term incubation (12 days, data not shown). Although it is not clear if the agonistic effect of the anti-HER2/new ScFv- TNF-α will enhance the malignancy of the ΗER2/neu expressing tumor cells in vivo, the potent immunomodulatory effect of the TNF-α could activate a potent immunity against HER2/rcew expressing tumor cells. [0144] In summary, we have constructed and characterized a novel anti-HER2Mew

ScFv fusion protein in which the ScFvs are trimerized by TNF-oc or TNF-Oc(S 147Y). These fusion proteins initiate robust HER2/πew signaling and, remarkably, facilitate the repair of the injured cultured epithelial cell monolayers. Unfortunately there is difficulty to evaluate the wound healing effect of anti-HER2/neu ScFv- TNF-α and anti-HER2/new ScFv- TNF- α(S147Y) in animal model or clinic. Although the human ΗER2/neu transgenic mice are available, it is not clear if the human ΗER2/neu is expressed appropriately on the epithelial cells. In addition, murine TNF-α moiety of anti-HER2/neu ScFv- TNF-α binds human TNF-α receptors, which may cause toxicity when administrated in vivo, and the immunogenicity of murine TNF-α also hampers the clinical use of anti-HER2/ne« ScFv- TNF-α and anti-ΗER2/neu ScFv- TNF-α(S147Y). An alternative approach will be to use the human TNF-α(Y87S) fused with the anti-HER2/ne« ScFv (anti-HER2/rcew ScFv- hTNF- α(Y87S)). With the human TNF-α lacking its biological activity but still maintaining the trimeric structure, anti-HER2/rce« ScFv- hTNF-α(Y87S) can be a therapeutic for wound healing in the clinic. Additionally, the general approach of using a mutant TNF-α to construct a polymeric antibody may be applicable to design a more effective multimeric vehicle.

[0145] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. AU publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

CLAIMSWhat is claimed is:

1. A composition for enhancing wound healing in a mammal, said composition comprising an anti-HER2/rcew antibody attached to a tumor necrosis factor alpha (TNF-α).

2. The composition of claim 1, wherein said antibody is a single chain antibody.

3. The composition of claim 2, wherein said antibody is a single chain Fv antibody (scFv).

4. The composition of claim 1, wherein the heavy chain of said antibody is a fusion protein with TNF-α, and the light chain of said antibody is covalently linked to the heavy chain.

5. The composition of claim 1, wherein the light chain of said antibody is a fusion protein with TNF-α, and the heavy chain of said antibody is covalently linked to the heavy chain.

6. The composition of any one of claims 4 or 5 wherein said the covalent linkage between the heavy and light chain of the antibody is via a disulfide linkage.

7. The composition of any one of claims 4 or 5 wherein the TNF-α and the antibody comprising the fusion protein are joined by a peptide linker.

8. The composition of claim 7, wherein the peptide linker is a (Gly₄Ser)₃ (SEQ ID NO: 5) linker.

9. The composition of claim 1, wherein said antibody is a C6 antibody.

10. The composition of claim 1, wherein said antibody comprises a C6MH3-B 1 variable heavy (VH) region.

11. The composition of claim 1, wherein said antibody comprises a C6MH3-B1 variable light (VL) region.

12. The composition of claim 10, wherein said antibody comprises a C6MH3-B1 variable light (VL) region.

13. The composition of claim 1, wherein said antibody is C6MH3-B1 scFv.

14. The composition of claim 1, wherein said antibody is joined to said TNF-α by a linker.

15. The composition of claim 1, wherein said antibody is joined to said TNF-α by peptide linker.

16. The composition of claim 1, wherein said antibody is joined to said TNF-α by (Gly₄Ser)₃ (SEQ ID NO:5) peptide linker.

17. The composition of claim 1, wherein said antibody joined to said TNF-α forms a fusion protein.

18. The composition of claim 1, wherein said TNF-α is a human TNF-α.

19. The composition of claim 1, wherein said TNF-α is modified human TNF-α having reduced or eliminated TNF-α activity, but retaining the ability to bind to two other TNF-α.

20. The composition of claim 19, wherein said TNF-α is a human TNF-α comprising the mutation Y87S.

21. The composition of claim 1, wherein said TNF-α is a murine TNF-α.

22. The composition of claim 21, wherein said TNF-α is a murine TNF-α comprising the mutation S 147 Y.

23. The composition of claim 1, wherein said anti-HER2/π<?« antibody attached to a tumor necrosis factor alpha (TNF-α) is a fusion protein comprising a C6MH3- Bl scFv attached to a human TNF-α comprising the mutation Y87S.

24. The composition of claim 23, wherein said scFv is attached to said TNF-α by a (Gly₄Ser)₃ (SEQ ID NO:5) linker.

25. The method of claim 1, wherein said composition comprises a complex consisting of three anti-HER2-TNFα.

26. The composition of claim 1, wherein said anti-ΗER2/neu antibody attached to a tumor necrosis factor alpha (TNF-α) is present in a pharmacologically acceptable excipient.

27. The method of claim 26, wherein said excipient is suitable for topical administration to the skin or eye.

28. The method of claim 26, wherein said composition is in a unit dosage formulation.

29. A method of enhancing wound healing in a mammal, said method comprising contacting a wounded tissue in said mammal with a composition according to any one of claims 1-28 in a dosage sufficient to enhance wound healing.

30. The method of claim 29, wherein said wound is selected from the group consisting of an acute wound, a chronic wound, a surgical wound, and an optical wound.

31. The method of claim 29, wherein said wound is selected from the group consisting of a wound to the skin, a wound to a mucosal surface, and a wound to an internal tissue or organ.

32. A dressing, wherein said dressing is impregnated with a composition according to any one of claims 1-28.

33. The dressing of claim 32, wherein said dressing is a sterile dressing.

34. A method of activating a HER2 receptor said method comprising contacting said HER2 receptor with a trimerized anti-HER2 antibody.

35. The method of claim 34, wherein said trimerized anti-HER2 antibody comprises a composition according to any one of claims 1-28.

36. A method of increasing Rac induced cell migration said method comprising contacting a tissue in a mammal with a trimerized anti-HER2 antibody.

37. The method of claim 36, wherein said trimerized anti-HER2 antibody comprises a composition according to any one of claims 1- 28.

38. A composition comprising a first moiety attached to a first TNF-α, a second moiety attached to a second TNF-α, and a third moiety attached to a third TNF-α, wherein said first, second and third TNF-α interact to form a trimer thereby coupling said first, second, and third moieties, and said first, second and third moieties are independently selected from the group consisting of an antibody, a ligand, an epitope tag, a cytokine, a growth factor, a receptor, a cytotoxin, a detectable label, a lipid, and a liposome.

39. A method of forming a trimeric complex of a first moiety, a second moiety, and a third moiety, said method comprising: providing said first moiety attached to a first TNF-α, said second moiety attached to a second TNF-α, and said third moiety attached to a third TNF-α; contacting the first, second, and third TNF-α with each other whereby said first, second and third TNFα interact to form a trimer thereby coupling said first, second, and third moieties to each other.

40. A method of enhancing wound healing, said method comprising contacting a wounded tissue with a polyvalent construct that specifically binds three or more EDER2/neu receptors.

41. A composition for enhancing wound healing in a mammal, said composition comprising at least three ΗER2/neu specific antibodies.

42. A kit for the enhancement of wound healing, said kit comprising: a container containing a composition according to any one of claims 1- 28.

43. The kit of claim 42, wherein said composition is provided in a dressing for a wound.

44. The kit of claim 43, wherein said dressing is a sterile dressing.

45. The kit of claim 42, further comprising instructional materials teaching the use of said composition to enhance wound healing and/or to reduce the formation of scan tissue and/or adhesions.