US20170260246A1

US20170260246A1 - Carcinoma homing peptide (chp), its analogs, and methods of using

Info

Publication number: US20170260246A1
Application number: US15/601,769
Authority: US
Inventors: Shulin Li; Jeffry Cutrera; Xueqing Xia
Original assignee: Louisiana State University and Agricultural and Mechanical College
Current assignee: Louisiana State University and Agricultural and Mechanical College
Priority date: 2011-02-11
Filing date: 2017-05-22
Publication date: 2017-09-14
Also published as: US9657077B2; US20120208770A1

Abstract

A mini-peptide and its analogs have been found to target gene products to tumors. The peptide, named Carcinoma Homing Peptide (CHP), increased the tumor accumulation of the reporter gene products in five independent tumor models, including one human xenogeneic model. A CHP-IL-12 fusion gene was also developed using CHP and the p40 subunit of IL-12. The product from CHP-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment. In three tumor models. CHP-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for CHP was identified as vimentin. CHP can be used to improve the efficacy and safety of targeted cancer treatments.

Description

The benefit of the Feb. 11, 2011 filing date of the U.S. provisional patent application Ser. No. 61/441,914 is claimed under 35 U.S. §119(e).
This invention was made with government support under grant number R01 CA120895 awarded by the National Institutes of Health. The government has certain rights in this invention.

TECHNICAL FIELD

This invention pertains to a carcinoma homing peptide and its analogs, compounds, and methods that target tumors, and methods to use these peptides including targeting, decreasing the size of, inhibiting growth of, and identification of mammalian tumors, such as breast adenocarcinoma, squamous cell carcinoma, and colon carcinoma.

BACKGROUND ART

The cytokine, interleukin 12 (IL-12), discovered by Giorgio Trinchieri in 1989 [1], bridges the innate and adaptive immune responses by inducing interferon-γ (IFN-γ) production primarily from natural killer and T cells. Cancer therapy with IL-12 exploits its natural immune functions to polarize T cells to the T _h1 phenotype, boost effector T cells, downregulate angiogenesis, remodel the extracellular matrix, and alter the levels of immune suppressive cytokines [2]. Due to these activities, IL-12 is one of the most promising cytokines for immunomodulatory cancer therapy.
The initial clinical trials with IL-12 resulted in grave toxicities including deaths, which severely downgraded the reputation and potential application of this effective cytokine. In reality, most anticancer drugs or biological modalities are associated with systemic toxicity. It is desirable to decrease this toxicity to effectively and safely treat the extremely high numbers of cancer patients [2].
A popular strategy for sequestering the effects of cytokine therapies in the tumor environment is targeting cellular markers that are upregulated exclusively in the tumor cells or the tumor microenvironment. Indeed, conjugating IL-12 to tumor-specific antibodies, such as L19 [3] and HER2 [4], and tumor vasculature-specific peptides, such as ROD [5] and CNGRC (SEQ ID NO:9) [6], improves the efficacy of treatments; however, the necessarily high frequency of administrations of recombinant cytokines increases the immunogenicity, toxicity, and cost of treatments. A gene therapy approach would reduce these limitations.
Intratumoral IL-12 gene therapy is able to eradicate 40% of tumors in a murine squamous cell carcinoma model (SCCVII) while systemic delivery via intramuscular administration fails to eradicate any tumors [7]; however, direct injection into tumor sites is rarely available noninvasively or post-surgically. Several methods have been developed to target the IL-12 effect to the tumor after systemic delivery. For example, modifying viral vectors with tissue specific gene promoters such as the CALC-I promoter [8], capsid-expressed tumor-specific peptides [9], and polyethylene glycol or other nanoparticles [10, 11] increases tumor specific expression and decreases systemic expression; however, the fenestrated vasculature of the tumor environment allows for the gene products to leak out of the tumor environment leading to systemic toxicities [12]. Therefore, a gene product that can interact with and remain in the tumor environment will increase the level of therapeutic efficacy and decrease systemic toxicity.
Tumor targeting can be achieved via the screening of various libraries to select tumor-targeted peptides, DNA/RNA aptamers, antibodies, etc; however, the only mechanism that can be used for homing gene products from systemically injected genes will be tumor-targeted mini-peptides encoding DNA. The small size of these peptides eliminates the concern of immunogenicity, and reduces the effect on the biological function of the gene product, though some minipeptidies may boost or inhibit gene function [20]. The tiny peptide encoding DNA sequences can be easily fused with any therapeutic gene. Finally, these peptides can complement existing tumor targeting approaches such as transcriptional targeting [8], translational targeting [21], and targeted delivery [3-6].
Currently, most tumor-targeting strategies are based on extremely specific interactions, and the ability to target the tumor environment is constrained to a single cell type or specific type of tumor. Proteins are conjugated with polyunsaturated fatty acids, monoclonal antibodies, folic acid, peptides, and several other chemicals to increase the tumor-targeted ability of the therapeutic protein. Other tumor targeting peptides can deliver small molecules with only one copy for each small-molecule payload but require multiple copies of the peptide to target larger molecules such as a full length cytokine [24].

DISCLOSURE OF THE INVENTION

We have discovered a new tumor targeting peptide, VNTANST (SEQ ID NO:1), and its analogs. A DNA fragment encoding VNTANST (SEQ ID NO:1) was inserted directly before the stop codon of the p40 subunit of the IL-12 encoding sequence in plasmid DNA. Transfection of this plasmid DNA via intramuscular (i.m.) electroporation (EP) into muscle tissue distal from the tumor site inhibited tumor growth and extended survival in multiple tumor models and two mouse strains and reduced lung metastasis in a spontaneous metastatic model. Due to this broad targeting nature and to simplify the description, the peptide VNTANST (SEQ ID NO:1) was renamed the Carcinoma Homing Peptide (CHP). We discovered that the linear peptide VNTANST (SEQ ID NO:1) increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type (wt) IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST (SEQ ID NO:1) was identified as vimentin, which is localized on the cell surface of tumor cells but not on normal cells. Vimentin expression in tumors is associated with the epithelial to mesenchymal transition and increased malignancy and metastasis in tumors. Lastly, this gene product-targeted approach minimized the risk of IL-12-induced toxicity. These results show the promise of using VNTANST (SEQ ID NO:1) to as a homing peptide to target therapeutic compounds to tumor cells, for example, to improve delivery of IL-12 treatments.
We have developed a fully functional tumor targeting IL-12 gene construct that can be delivered systemically for treating distally located neoplastic diseases. We have administered the peptide CHP-IL-12 by direct intravenous injection, and have directly injected the gene construct into tissue followed by electroporation. Inserting peptide-encoding sequences directly prior to the stop codon in the p40 gene of an IL-12 plasmid did not interfere with transcription, translation, post-translational modifications, or therapeutic functionality of the IL-12 gene product. Also, CHP maintained its tumor-targeting ability as seen in IL-12^−/− mice and increased the therapeutic efficacy of systemic IL-12 gene-therapy treatments while decreasing liver toxicity. In fact, CHP-IL-12 may home or target the tumor better than CHP alone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts the peptide-SEAP (secreted alkaline phosphatase) constructs with insertion of the peptide-coding sequence directly before the stop codon (arrow). CMV shows the location of the cytomegalovirus promoter; IVS shows the location of the intron; pA shows the location of the bovine growth hormone polyadenylation signal; SEAP shows the location of the secreted alkaline phosphatase-coding sequence; STOP shows the location of the stop codon.

FIG. 1B shows TIS SEAP (ratio of the SEAP activity between tumors and serum) levels 72 hours after i.m. EP of several peptide-SEAP plasmid DNAs in syngeneic CT26 (n=3), SCCVII (n=4), AT84 (n=4), and 4T1 (n=4) tumor-bearing mice, as well as xenogeneic MCF7 (n=4) tumor-bearing mice.

FIG. 1C shows DAB (diaminobenzidine) staining of tumor tissues from CHP-biotin treated mice counterstained with either hematoxylin (left) or eosin (right). The bottom images are larger versions of the areas within the white squares. Bar=100 μm in the top panels and bar=200 μm in the bottom panels.

FIG. 1D shows DAB staining of tumor tissues from Control-peptide-biotin treated mice counterstained with either hematoxylin (left) or eosin (right). The bottom images are larger versions of the areas within the white squares. Bar=100 μm in the top panels and bar=200 μm in the bottom panels.

FIG. 2A depicts the CHP-IL-12 construct with insertion of the CHP-coding sequence directly before the stop codon in the p40 subunit of IL-12 (arrow). CMV shows the location of the cytomegalovirus promoter; IVS shows the location of the intron; SEAP shows the location of the SEAP-coding sequence; STOP shows the location of the stop codon; and, pA shows the location of the bovine growth hormone polyadenylation signal.

FIG. 2B shows expression of IL-12 after in vitro transfection of 4T1 cells with control, wtIL-12, CDGRC-IL-12, and CHP-IL-12 (n=3).

FIG. 2C shows induction of IFN-γ from splenocytes after transfer of condition medium containing Control, wtIL-12, CDGRC-II-12, or CHP-IL-12 gene products.

FIG. 2D shows IL-12 accumulation in tumor-bearing IL-12^−/− mice treated with CHP-IL-12 or wtIL-12 determined via an IL-12p70 ELISA. Columns represent the wtIL-12-normalized level of IL-12/protein (pg/mg) in tumor per IL-12/protein (pg/mg) in kidneys, livers, and spleens and IL-12 μg/mL serum (n=4). Error bars represent the standard error of the mean (SEM) (*represent p<0.05 compared to all groups).

FIG. 3A shows tumor growth following treatments with CHP-IL-12, wtIL-12, and control plasmid DNA in 4T1 tumor-bearing balb/c mice (n=5; *represents p<0.05 at day 30 and p<0.001 from day 33 until day 42 compared to wtIL-12 plasmid DNA and p<0.01 at day 21 and p<0.001 from day 24 to day 33 compared to control plasmid DNA).

FIG. 3B shows metastatic nodules in the lungs of 4T1 tumor-bearing balb/c mice (n=5) treated with CHP-IL-12, wtIL-12, and control plasmid DNA and sacrificed 17 days after the second treatment (*represents p<0.05 compared to wtIL-12 plasmid DNA; # represents p<0.001 compared to control plasmid DNA).

FIG. 3C shows Kaplan-Meier survival analysis of the 4T1 tumor-bearing balb/c mice treated with CHP-IL-12, wtIL-12, and control plasmid DNA (*represents p<0.05 compared to wtIL-12 plasmid DNA; # represents p<0.001 compared to control plasmid DNA).

FIG. 3D shows tumor growth following treatments with CHP-IL-12, wtIL-12, and control plasmid DNA in SCCVII tumor-bearing C3H mice (n=5; *represents p<0.05 on

days

17 and 20 compared to wtIL-12 plasmid DNA and control plasmid DNA).

FIG. 3E shows Kaplan-Meier survival analysis of the SCCVII tumor-bearing C3H mice treated with CHP-IL-12, wtIL-12, and control plasmid DNA (*represents p<0.05 compared to wtIL-12 and control plasmid DNA).

FIG. 3F shows tumor growth following treatments with CHP-IL-12, wtIL-12, and control plasmid DNA in CT26 tumor-bearing balb/c mice (n=5; *represents p<0.05 compared to wtIL-12 plasmid DNA, n=4, on day 25, and control plasmid DNA, n=3, on days 19 through 25). Black arrows represent treatments, and error bars represent SEM.

FIG. 4A shows fluorescence-activated cell sorting (FACS) analysis of tumor infiltrating cells isolated from SCCVII tumors from C3H mice following intravenous (i.v.) injection of Control, wtIL-12, or CHP-II-12, with or without depletion of vimentin with a co-injection of purified polyclonal goat anti-vimentin (100 μg) in the same i.v. injection as the peptide-biotin collected 7 days after the second treatment. The top right quadrant of the dot plot representation of cells gated for CD11c⁺ represents activated DC (CD81^hi).

FIG. 4B shows tumor-specific cytotoxic T lymphocyte (CTL) activity from wtIL-12 and CHP-IL-12 fusion gene plasmid DNA treated mice bearing orthotopic EMT6 (a transplantable mouse mammary tumor cell line) tumors collected (*represents p<0.05).

FIG. 4C shows serum IFN-γ levels from 4T1-tumor bearing Balb/c 3 days after treatments with CHP-IL-12, wtIL-12, and control plasmid. Error bars represent SEM.

FIG. 5A shows SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis of potential receptors for CHP isolated via affinity chromatography of a pool of cell-surface proteins isolated from SCCVII cells. The only distinct band (arrow) was located in the second fraction, and mass spectrometry identified this band as vimentin. “BSA” represents bovine serum albumin.

FIG. 5B shows the interaction of CHP-biotin with recombinant vimentin-GST (Vimentin), GST, and coating buffer only (control) coated wells of a polystyrene plate (n=6; *represents p<0.001 compared to both GST and Control, errors bars represent SEM).

FIG. 5C shows Western blot analysis of vimentin expression in an SCCVII tumor (1) and heart (2), lung (3), liver (4), kidney (5), spleen (6), and serum (7) from SCCVII-tumor bearing C3H mice. “GAPDH” represents glyceraldehyde 3-phosphate dehydrogenase.

FIG. 5D shows Western blot analysis of vimentin expression in in vitro and ex vivo tumor samples from SCCVII, CT26, 4T-1, and B16F10 tumors.

FIG. 5E shows accumulation of peptide-biotin in syngeneic SCCVII tumor bearing C3H mice following i.v. injection of either Control-biotin (top left and right) or CHP-biotin (bottom left and right), with (top and bottom right) or without (top and bottom left) depletion of vimentin with a co-injection of purified polyclonal goat anti-vimentin (100 μg) in the same i.v. injection as the peptide-biotin.

FIG. 6A shows the number of SCCVII tumor-bearing C3H mice with toxic lesions on the liver following two treatments of 1 μg (2×1 μg), 2 μg (2×2 μg), or 10 μg (2×10 μg) or three treatments of 2 μg (3×2 μg) of wtIL-12 or CHP-IL-12 (n=12).

FIG. 6B shows a representative image of a normal liver area from the SCCVII tumor-bearing C3H mice. Scale bar represents 50 μm.

FIG. 6C shows a representative image of a toxic lesion from the SCCVII tumor-bearing C3H mice. Scale bar represents 50 μm.

FIG. 6D shows levels of alanine transaminase (ALT), a key indicator of liver function, for both plasmid DNA treatments (wtIL-12 and CHP-IL-12) at all DNA levels and difference time points.

FIG. 7A shows SEAP activities in the tumors of the same CT26-tumor bearing mice used in FIG. 1B after peptide-SEAP plasmid DNA intramuscular electroporation of several peptides.

FIG. 7B shows SEAP activities in the serum of the same CT26-tumor bearing mice used in FIG. 1B after peptide-SEAP plasmid DNA intramuscular electroporation of several peptides.

FIG. 8 shows sections from the hearts, lungs, livers, kidneys, and spleens from the same mice in FIG. 2B, FIG. 2C and FIG. 2D, counterstained with eosin only.

FIG. 9 shows the level of CHP-specific IgG from EMT6-tumor bearing Balb/c mice treated with wtIL-12 or CHP-IL-12 gene therapy as determined via binding to wells of a microwell plate coated with coating buffer only, control peptide or CHP peptide (n=3).

FIG. 10 shows the activity of CHP-SEAP when bound to vimentin. The induction of IFN-γ from splenocytes by CHP-IL12 and wtIL12 was compared when in the presence of vimentin or BSA. Error bars represent SEM and n=3.

FIG. 11 shows the tumor volume in SCCVII tumor-bearing C3H mice at various days after inoculation with various gene constructs, each comprising the named peptide added to the p40 subunit of IL-12 prior to the stop codon.

MODES FOR CARRYING OUT THE INVENTION

Tumor targeting can be achieved via the screening of various libraries to select tumor-targeted peptides, DNA/RNA aptamers, antibodies, and other known strategies. However, the only mechanism that can be used for homing gene products from systemically injected genes is the use of DNA sequences encoding for tumor-targeted mini-peptides. The small size of these peptides eliminates the concern of immunogenicity, as shown below, and reduces the effect on the biological function of the gene product, though some mini-peptides may boost or inhibit gene function [20]. The peptide-encoding DNA sequences can be easily fused with any therapeutic gene. Finally, the use of the mini-peptides can complement existing tumor targeting approaches such as transcriptional targeting, translational targeting, and targeted delivery].
We have discovered a tumor-targeting 7-amino-acid peptide, carcinoma homing peptide (“CHP,” amino acid sequence of VNTANST (SEQ ID NO: 1)). The peptide VNTANST (SEQ ID NO:1) was previously reported to target normal lungs when present on the surface of virus particles [14]. We have shown that CHP was more effective than the known cyclic tumor-homing peptides such as CNGRC (SEQ ID NO:9) and RGD4C for targeting to tumors, which rely on disulfide bonds to maintain the cyclic structure of the targeting peptides.
Other tumor targeting peptides have been shown to deliver small molecules with only one copy for each small-molecule payload but require multiple copies of the peptide to target larger molecules such as a full length cytokine [24]. We have shown that fusion of a single copy of CHP-encoding DNA (gtcaacacggctaactcgaca (SEQ ID NO:2)) with the p40 subunit of IL-12 boosted the accumulation of IL-12 in tumors, suggesting one copy of CHP is sufficient to carry one copy of IL-12 to the tumor site.
Currently, most tumor-targeting strategies are based on extremely specific interactions, and the ability to target the tumor environment is constrained to a single cell type or specific type of tumor. We have shown, as discussed below, that CHP increased the efficacy of IL-12 gene therapy to inhibit tumor growth in the three tumor cell lines (i.e., breast adenocarcinoma, squamous cell carcinoma, and colon carcinoma), and in two different mouse strains. In addition, CHP-IL-12 extended survival more than wtIL-12 treatments in both the breast adenocarcinoma and squamous cell carcinoma cell lines. Similarly, CHP-IL-12 treatments inhibited the development of spontaneous lung metastasis, which is the primary killer of cancer patients. This increase in anti-tumor response was associated with increases in both tumor-specific cytotoxic T lymphocyte (CTL) activity and IL-12 accumulation in tumors. This result was in agreement with the result that intratumoral delivery of IL-12 yields better anti-tumor efficacy than systemic delivery [7]. The discovery of CHP is important since it will allow for systemic delivery to target IL-12 to tumors without the need of intratumoral delivery, which is not realistic for treating internal tumors, metastatic tumors, and residual tumor cells after standard therapy.
We also identified vimentin as a cell receptor for CHP. Vimentin is an intermediate filament protein conventionally regarded as an intracellular structural protein in cells of mesenchymal origin such as fibroblasts, chondrocytes, and macrophages [15]. Vimentin expression has been reported to be increased in several tumor models, including human prostate, colon [17], hepatocellular [16], and gemcitabine-resistant pancreatic cancers[19], and the tumor stromal cells in human colorectal tumors [18]. The upregulation of vimentin is associated with the epithelial-to-mesenchymal transition (EMT), which is important for motility as well as metastasis in several tumors. In addition, vimentin was recently discovered to be expressed on the cell surface of tumor cells [25] and epithelial cells during angiogenesis [26]. Additionally, some human tumor-initiating cells remaining after treatment overexpress vimentin on the tumor cell surface [27]. Another important aspect of vimentin is the conserved sequences among mouse, rat, dog, and humans [28]. This information along with our result for the tumor/serum SEAP accumulation in the xenogeneic human tumor model indicates that CHP targeting will be effective in human treatments.
We also confirmed (as discussed below) that vimentin is expressed at very low levels in the heart, liver, kidney, spleen, and serum of C3H mice, yet it is highly expressed in lung tissue. However, since most general expression of vimentin is intracellular [15, 29, 30], this expression should not be a target of CHP. We found that there was no accumulation of CHP-biotin in the lung sections which supports this theory. Conversely, as shown below, vimentin is highly expressed in aggressive murine squamous cell carcinoma (SCCVII) tumors in C3H mice, and CHP-biotin accumulated in the SCCVII tumors. Likewise, the tumor cells and corresponding syngeneic tumors both expressed detectable levels of vimentin. The differences seen between expression in tumor cell lines and the respective tumor tissues was due to the heterogeneous nature and multiple cell types in the tumor microenvironment.
We have developed a fully functional tumor-targeting IL-12 p40 gene construct based on CHP that can be delivered systemically for treating distally located neoplastic diseases. Inserting peptide-encoding sequences directly prior to the stop codon in the p40 subunit gene of an IL-12 plasmid did not interfere with transcription, translation, post-translational modifications, or therapeutic functionality of the IL-12 gene product. Also, CHP maintained its tumor-targeting ability as seen in IL-12−/− mice and increased the therapeutic efficacy of systemic IL-12 gene-therapy treatments, while decreasing liver toxicity. CHP-IL-12 was found to be more effective in decreasing tumor growth than other mini-peptides linked to the same p40 subunit of IL-12.
The term “CHP” used herein and in the claims refers to the peptide VNTANST. The term “CHP analogs” is understood to be peptides with consecutive sequences of 3 or more amino acids from VNTANST (SEQ ID NO:1) and that exhibit a qualitatively similar effect to the unmodified VNTANST (SEQ ID NO:1) peptide. Based on the effective size of other mini-peptides, we believe that effective CHP analogs include any three or greater consecutive amino acid sequence found within the CHP sequence, more preferably any four or greater consecutive amino acid sequence found within the CHP sequence, and most preferable any five or six consecutive amino acid sequence found within the CHP sequence. In addition, any DNA sequence that codes for any of the above VNTANST (SEQ ID NO:1) sequence or CHP analog sequences can be used for making tumor targeting constructs. In the experiments below, we used the DNA sequence of gtcaacacggctaactcgaca (SEQ ID NO:2) to encode for CHP, but due to the degeneracy of the DNA code, any DNA sequence that would code for CHP could be used. In addition, any DNA sequence that encodes for the CHP analogs could be used. CHP or CHP analog may be a synthetic or recombinant peptide. With its specific tumor targeting property, CHP peptide or CHP analogs or the DNA encoding for CHP or CHP analogs can carry therapeutic proteins, peptides, drugs, genes, cells, viral or nonviral vectors, bacteria and other modalities into tumor tissues, reducing the toxicity to other organs and increasing the therapeutic efficacy. As a result, a low dose of the peptide or construct may be needed for treating tumors. CHP or CHP analogs or the corresponding DNA encoding for CHP or CHP analogs can also be used to carry therapeutic agents for prevention or treatment of metastatic tumors. Therapeutic agents are well known in the art (e.g., peptides, chemotherapeutic agents, liposomes, nanoparticles) that can be conjugated to a targeted peptide for increased accumulation of the therapeutic agent in the tumor environment.
CHP and CHP analogs can be used in a variety of applications including exploratory studies to diagnose tumors or tumor metastasis in combination with image tools, to monitor the effect of treatments in combination with image tools, and to deliver therapeutic agents for treating metastatic tumors and tumors localized in internal organs as well as prevent tumor recurrence from residual tumors after standard therapy. The therapeutic agents to be carried by CHP and CHP analogs include anti-tumor drugs, peptides, proteins, genes, cells, viral/nonviral vectors, bacteria and others. For example, the p40 subunit of the protein IL-12 was used below. We have made a new conjugate of CHP and the p40 subunit of IL-12. The sequence of this new construct is found in Table 1, below. The peptide sequence for CHP-IL-12 is SEQ ID NO: 3, and the nucleic acid sequence is SEQ ID NO: 4. Initial work on conjugating other cytokines to CHP, for example IL-15 and PF4, indicate that some increase in efficacy was seen for IL-15, but that in these initial tests, no increase in efficacy was seen in CHP-PF4.
CHP and CHP analogs can be administered by methods known in the art. In our work, we have used both direct injection of the gene construct into tissue followed by electroporation, and have directly injected the peptide intravenously. As a DNA gene construct, the delivery can be from vectors which may be derived from viruses or from bacterial plasmids. There are many methods to deliver gene constructs to tumors or targeted tissues. Some examples of the various delivery systems can be found in U.S. Pat. Nos: 5,910,488; 7,192,927; and 7,318,919; whose descriptions of such delivery systems are hereby incorporated by reference. In addition, the vector delivery system may incorporate a promoter sequence to initiate transcription of the gene construct.

Claims

1. A tumor-targeting conjugate comprising an agent conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1.

2. The tumor-targeting conjugate of claim 1, wherein the agent is a reporter peptide or a protein tag or an antitumor therapeutic agent.

3. The tumor-targeting conjugate of claim 2, wherein the reporter protein is secreted alkaline phosphatase.

4. The tumor-targeting conjugate of claim 2, wherein the protein tag is biotin.

5. The tumor-targeting conjugate of claim 2, wherein the anti-tumor therapeutic agent is a cytokine.

6. A composition comprising the tumor targeting conjugate of claim 1.

7. The tumor-targeting conjugate of claim 1, prepared by a method comprising conjugating the agent to the carcinoma homing peptide (CHP) consisting of SEQ ID NO:1.

8. A method for targeting an agent to a vimentin-expressing cell comprising contacting the vimentin-expressing cell with a tumor targeting conjugate comprising the agent conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1,

wherein the CHP binds to the vimentin.

9. The method of claim 8, wherein the agent is an anti-tumor therapeutic agent.

10. The method of claim 9, wherein the anti-tumor therapeutic agent is a cytokine.

11. The method of claim 9, wherein the cytokine is interleukin 12.

12. The method of claim 9, wherein the anti-tumor therapeutic agent is a p40 subunit of interleukin 12.

13. The method of claim 8, wherein the tumor targeting conjugate comprises an amino acid sequence of SEQ ID NO:3.

14. The method of claim 8, wherein the tumor targeting conjugate is encoded by a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:2.

15. A method of determining the presence of a vimentin protein on a surface of a cell comprising:

contacting the cell with a tumor targeting conjugate comprising a peptide conjugated to a carcinoma homing peptide (CHP) consisting of SEQ ID NO:1 wherein the tumor targeting conjugate binds to the vimentin protein; and

assaying the cell for the presence of a bound tumor targeting conjugate such that the presence of the bound tumor targeting conjugate correlates with the presence of the vimentin protein on the cell.

16. The method of claim 15, wherein the first peptide is a reporter peptide or a protein tag.

17. The method of claim 16, wherein the reporter peptide is secreted alkaline phosphatase.

18. The method of claim 16, wherein the protein tag is biotin.

19. The method of claim 15, wherein the cell is from a biological sample from a mammal.

20. The method of claim 19, further comprising

determining an amount of the vimentin protein present on the cell; and

comparing the amount of the vimentin protein present on the cell to a control amount of vimentin protein present on a control cell,

wherein the presence of an increased amount of the vimentin protein present on the cell as compared with the control amount indicates that the cell is cancerous.