# Patent

## Title
DNA composition encoding an immunogenic VEGF receptor peptide and methods of use thereof

## Abstract

A method of inhibiting endothelial cell proliferation in a mammal is provided. The method comprises the step of administering to the mammal an effective immunological response eliciting amount of a DNA composition comprising a DNA construct operably encoding a VEGF receptor polypeptide and a pharmaceutically acceptable carrier therefor, whereby said mammal exhibits an immune response elicited by vaccine and specific to proliferating endothelial cells. The methods of this invention inhibit vascular endothelial cell proliferation in the tumor micro-environment. Angiogenesis inhibition and subsequent decrease in tumor growth and dissemination is achieved.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. Application Ser. No. 11/507,298, filed on Aug. 21, 2006, now U.S. Pat. No. 8,048,428, which is a continuation-in-part of U.S. application Ser. No. 10/090,183, filed on Mar. 2, 2002, now U.S. Pat. No. 7,094,410, each of which is incorporated herein by reference in its entirety.


GOVERNMENTAL RIGHTS
A portion of the work described herein was supported by grant numbers 5-70373-COLON, CA83856, and CA 11571-A1 from the National Institutes of Health, as well as grant numbers DAMD17-02-01-0137 and DAMD17-02-01-0562 from the Department of Defense. The United States Government has certain rights in this invention.


FIELD OF THE INVENTION
This invention relates to deoxyribonucleic acid (DNA) compositions encoding peptides effective for eliciting an immune response against proliferating endothelial cells. More particularly this invention relates to DNA compositions encoding for an immunogenic vascular endothelial growth factor (VEGF) receptor or an immunogenic fragment thereof. This invention also relates to methods of using the DNA composition to inhibit vascular endothelial cell proliferation, tumor growth, and angiogenesis.
BACKGROUND OF THE INVENTION
Vaccines have been utilized to provide a long term protection against a number of disease conditions by very limited administration of a prophylactic agent that stimulates an organism's immune system to destroy disease pathogens before they can proliferate and cause a pathological effect. Various approaches to vaccines and vaccinations are described in Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology, Principles and Applications of Recombinant DNA, Second Edition, ASM Press pp. 253-276 (1998).
Vaccination is a means of inducing the body's own immune system to seek out and destroy an infecting agent before it causes a pathological response. Typically, vaccines are either live, but attenuated, infectious agents (virus or bacteria) or a killed form of the agent. A vaccine consisting of a live bacteria or virus must be non-pathogenic. Typically, a bacterial or viral culture is attenuated (weakened) by physical or chemical treatment. Although the agent is nonvirulent, it can still elicit an immune response in a subject treated with the vaccine.
An immune response is elicited by antigens, either specific macromolecules, or an infectious agent. These antigens are generally either proteins, polysaccharides, lipids, or glycolipids, which are recognized as “foreign” by lymphocytes known as B cells and T cells. Exposure of both types of lymphocytes to an antigen elicits a rapid cell division and differentiation response, resulting in the formation of clones of the exposed lymphocytes. B cells produce plasma cells, which in turn, produce proteins called antibodies (Ab), which selectively bind to the antigens present on the infectious agent, thus neutralizing or inactivating the pathogen (humoral immunity). In some cases, B cell response requires the assistance of CD4 helper T cells.
The specialized T cell clone that forms in response to the antigen exposure is a cytotoxic T lymphocyte (CTL), which is capable of binding to and eliminating pathogens and tissues that present the antigen (cell-mediated or cellular immunity). In some cases, an antigen presenting cell (APC) such as a dendritic cell, will envelop a pathogen or other foreign cell by endocytosis. The APC then processes the antigens from the cells, and presents these antigens in the form of a histocompatibility molecule:peptide complex to the T cell receptor (TCR) on CTLs, thus stimulating an immune response.
Humoral immunity characterized by the formation of specific antibodies is generally most effective against acute bacterial infections and repeat infections from viruses, whereas cell-mediated immunity is most effective against viral infection, chronic intracellular bacterial infection, and fungal infection. Cellular immunity is also known to protect against cancers and is responsible for rejection of organ transplants.
Antibodies to antigens from prior infections remain detectable in the blood for very long periods of time, thus affording a means of determining prior exposure to a pathogen. Upon re-exposure to the same pathogen, the immune system effectively prevents reinfection by eliminating the pathogenic agent before it can proliferate and produce a pathogenic response.
The same immune response that would be elicited by a pathogen can also sometimes be produced by a non-pathogenic agent that presents the same antigen as the pathogen. In this manner, the subject can be protected against subsequent exposure to the pathogen without having previously fought off an infection.
Not all infectious agents can be readily cultured and inactivated, as is required for vaccine formation, however. Modern recombinant DNA techniques have allowed the engineering of new vaccines to seek to overcome this limitation. Infectious agents can be created that lack the pathogenic genes, thus allowing a live, nonvirulent form of the organism to be used as a vaccine. It is also possible to engineer a relatively nonpathogenic organism such as E. coli to present the cell surface antigens of a pathogenic carrier. The immune system of a subject treated with such a transformed carrier is “tricked” into forming antibodies to the pathogen. The antigenic proteins of a pathogenic agent can be engineered and expressed in a nonpathogenic species and the antigenic proteins can be isolated and purified to produce a “subunit vaccine.” Subunit vaccines have the advantage of being stable, safe, and chemically well defined; however, their production can be cost prohibitive.
A new approach to immunization has emerged in recent years, broadly termed genetic immunization. In this approach, a gene encoding an antigen of a pathogenic agent is operably inserted into cells in the subject to be immunized. The treated cells are transformed and produce the antigenic proteins of the pathogen. These in vivo-produced antigens then trigger the desired immune response in the host. The genetic material utilized in such genetic vaccines can be either a DNA or RNA construct. Often the polynucleotide encoding the antigen is introduced in combination with other promoter polynucleotide sequences to enhance insertion, replication, or expression of the gene.
DNA compositions encoding antigens can be introduced into the host cells of the subject by a variety of expression systems. These expression systems include prokaryotic, mammalian, and yeast expression systems. For example, one approach is to utilize a viral vector, such as vaccinia virus incorporating the new genetic material, to innoculate the host cells. Alternatively, the genetic material can be incorporated in a vector or can be delivered directly to the host cells as a “naked” polynucleotide, i.e. simply as purified DNA. In addition, the DNA can be stably transfected into attenuated bacteria such as Salmonella typhimurium. When a patient is orally vaccinated with the transformed Salmonella, the bacteria are transported to Peyer's patches in the gut (i.e., secondary lymphoid tissues), which then stimulate an immune response.
DNA compositions encoding antigens provide an opportunity to immunize against disease states that are not caused by traditional pathogens, such as genetic diseases and cancer. Typically, in a genetic cancer vaccine, antigens to a specific type of tumor cell must be isolated and then introduced into the vaccine. An effective general vaccine against a number of cancers can thus entail development of numerous individual vaccines for each type of cancer cell to be immunized against.
One general approach to treatment of tumors involves administering angiogenesis inhibiting compounds to patients with growing tumors. Angiogenesis is the process by which new capillaries and blood vessels form. Angiogenesis is important in embryonic development, tissue growth, tissue repair, and tissue regeneration. In addition to these normal and essential processes, angiogenesis is also involved in many abnormal pathological processes such as tumor growth, tumor metastasis, and ocular vascular diseases such as diabetic retinopathy.
Angiogenesis involves a number of interdependent processes, including (a) activation of vascular endothelial cells, (b) decomposition of cell matrix proteins by endothelial cells expressing protease activity, (c) migration of endothelial cells to a potential growth sites, (d) proliferation of endothelial cells and (e) tube formation by differentiation of endothelial cells. Each of these processes is affected by a variety of promoter substances such as fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factors (VEGF). The vascular endothelial growth factors (collectively VEGF) play a crucial role in endothelial cell growth and differentiation. VEGF acts by binding to receptor protein-tyrosine kinases present in the endothelial cell membranes, which in turn initiate a cascade of signal transduction reactions that stimulate cell growth.
Inhibition of pathological angiogenesis has been proposed as a treatment for tumors. See, for example, Folkman et al. Science, 221, 719, (1983). The basic concept of such treatment is that, since tumors require vascularization to grow, inhibition of blood vessel formation, through the administration of angiogenesis inhibiting compounds, will prevent tumor growth by starving the tumor of its blood supply. A disadvantage of his approach is that angiogenesis inhibitors must be administered on a relatively continuous basis to prevent tumor growth. A cessation in delivery of the inhibitor can lead to a resumption of tumor growth. A DNA composition effective for eliciting an antiangiogenic immune response would be an attractive preventative agent against tumor formation.
There is a continuing need for a generally effective compositions for inhibiting angiogenesis and growth of a variety of tumors without the need for targeting specific tumor antigens. The present invention satisfies this need.
SUMMARY OF THE INVENTION
A DNA composition effective for inhibiting endothelial cell proliferation comprises a DNA construct that encodes a VEGF receptor polypeptide, which can be a full length VEGF receptor or an immunogenic fragment of a VEGF receptor, which is capable of eliciting an immune response against vascular endothelial cells, is expressible in immune cells, and is incorporated in a pharmaceutically acceptable carrier. VEGF receptors include VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6; the murine homolog of KDR), e.g., DNA sequences set forth in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO 5, respectively. Preferably, immunogenic fragments of a VEGF receptor consist of about 8 to about 10 consecutive amino acid residues of a VEGF receptor.
The DNA compositions of the invention can encode a single immunogenic fragment of the VEGF receptor, a plurality of immunogenic fragments of the VEGF receptor in the form of a “minigene.”, or an entire VEGF receptor. When the compositions encode two or more immunogenic fragments of a VEGF receptor, one fragment preferably is linked to another fragment in a linear manner, i.e., by a spacer peptide.
The DNA composition can comprise a linear nucleic acid such as a purified DNA construct, or a DNA construct incorporated in a plasmid vector. Preferably, the DNA construct is incorporated in an attenuated bacterial or viral vector in a manner such that the polypeptide encoded by the construct will be expressed in immune cells (e.g., macrophages and/or dendritic cells) within the subject to which the DNA composition has been administered. In this manner, the DNA compositions of the present invention stimulate formation of cytotoxic T lymphocytes (CTLs) active against proliferating endothelial cells that overexpress a VEGF receptor.
Endothelial cells form the lining of mammalian vascular tissue. The proliferation of endothelial cells is a key process in angiogenesis. The compositions of the present invention provide a method for producing long term inhibition of angiogenesis in an organism treated with the composition by eliciting an immune response against proliferating endothelial cells. Non-proliferating endothelial cells, such as the linings of established blood vessels, do not present significant quantities of VEGF receptor antigens, and thus remain substantially unaffected by the CTLs that are produced in response to the DNA compositions.
In a method aspect of the present invention, a DNA composition is utilized to provide long term inhibition of endothelial cell proliferation in a treated patient. In one method embodiment, a DNA composition (vaccine) comprising a polynucleotide construct encoding a VEGF receptor polypeptide is administered orally to a patient in need of inhibition of endothelial cell proliferation in an amount that is sufficient to elicit an immune response against proliferating endothelial cells.
The present invention also provides a method of inhibiting angiogenesis in a patient treated with a DNA composition of the invention. In such a method embodiment, an immune response eliciting amount of a DNA composition that includes a DNA construct encoding a VEGF receptor polypeptide is administered to a patient suffering from an angiogenesis-related disease.
In yet another method aspect of the present invention, tumor growth is inhibited by treating a patient with a DNA composition of the invention. In such a method embodiment, an immune response-eliciting amount of a DNA composition comprising a DNA construct operably encoding a VEGF receptor polypeptide is administered to a patient having a growing tumor. Administration of the composition results in tumor growth arrest. Destruction of proliferating endothelial cells by the patient's immune system prevents vascularization of the tumor, in essence starving the tumor to death.
In the method embodiments of the present invention, the DNA compositions can be administered enterally, such as by oral administration, or perenterally, such as by injection or intravenous infusion. The VEGF receptor polypeptide can be a full length VEGF receptor or an immunogenic fragment thereof.
The compositions of the present invention are useful for treatment and prevention of a number of disease states. For example, a patient suffering from a cancer, diabetic retinopathy, and the like, can benefit from immunization by the compositions of the present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
In the Drawings,
FIG. 1 depicts the DNA sequence encoding human KDR, SEQ ID NO: 1.
FIG. 2 depicts the protein sequence of human KDR, SEQ ID NO: 2.
FIG. 3 depicts the DNA sequence encoding human Flt-1, SEQ ID NO: 3.
FIG. 4 depicts the protein sequence of human Flt-1, SEQ ID NO: 4.
FIG. 5 depicts the DNA sequence encoding mouse Flk-1, SEQ ID NO: 5.
FIG. 6 depicts the protein sequence of human Flk-1, SEQ ID NO: 6.
FIG. 7 is a pictorial representation of mouse lungs having varying levels of tumor coverage ranging from >50% coverage (labeled 3) to <10% coverage (labeled 1).
FIG. 8 is a graphical representation of data demonstrating that mice treated with a DNA composition encoding a VEGFR polypeptide (solid, heavy black line) and challenged by intravenous injection of CT-26 colon carcinoma cells, exhibited significantly reduced mortality relative to two control groups of mice (naive mice: solid thin line; control: dash-dot line).
FIG. 9 is a graphical representation of data demonstrating the suppression of D121 Lewis lung carcinoma tumor growth in mice treated with a DNA composition encoding a VEGFR polypeptide (pcDNA3.1-FLK-1) relative to two control groups of mice.
FIG. 10 is a graphical representation of data demonstrating the suppression of B16 melanoma tumor growth in mice vaccinated with a DNA composition encoding a VEGFR protein (●) relative to a control group (◯).
FIG. 11 is a graphical representation of data demonstrating the upregulation of CD25, CD69, and CD2 positive CD8+ T cells in mice treated with a DNA composition encoding a VEGFR polypeptide relative to a control group of mice.
FIG. 12. (A) Schematic map: Minigenes encoding the murine H-2 Dd- and Kd-restricted Flk-1 nonapeptides and spacers were assembled by PCR with overlapping oligonucleotides as templates. The PCR fragments generated were cloned downstream of ubiquitin in a modified pcDNA expression vector by using Xho I and Xba I restriction sites. (B) Proteins encoded by minigenes were expressed in mammalian cells. 293 T cells were transfected separately with either pDd-myc, pDd+Kd1-myc, pKd-myc or pKd+Dd2-myc for 24 hours, harvested, lysed and analyzed by Western blotting with anti-myc monoclonal antibody.
FIG. 13. Top panel: Schematic of experimental protocol. Lower panel: Survival rate of mice 25 days after tumor cell challenge. The Flk-1 whole gene vaccine was used as positive control. Groups of BALB/c mice (n=8) were immunized 3 times at 1 week intervals with attenuated Salmonella typhimurium RE-88 harboring the vectors indicated. Mice were challenged 2 weeks after the last immunization by i.v. injection of 2×105 D2F2 breast carcinoma cells.
FIG. 14. Top panel: Schematic of experimental protocol. Lower panel: Average lung weights of mice from each experimental group 19 days after tumor cell challenge. The average lung weight of normal mice was about 0.2 g. The Flk-1 whole gene vaccine was used as positive control. Groups of BALB/c mice (n=8) were immunized 3 times at 1 week intervals with attenuated Salmonella typhimurium harboring the vectors indicated. Mice were challenged 2 weeks after the last immunization by i.v. injection of 1×105 CT-26 colon carcinoma cells. CD8 depletion was performed by i.p. injections of anti-CD8 antibody (2.43, 0.5 mg/mouse) 1 day before tumor challenge, and repeated weekly injections.
FIG. 15. (A) Surface expression of Flk-1 by the endothelial cell line HEVc and by HEVc cells stably transfected with Flk-1 (HEVc-Flk). Cells were washed and incubated with PE-conjugated isotype control Ab (thin grey lines), or PE-conjugated anti-Flk-1 Ab (thick black lines). Groups of immunized BALB/c mice (n=4) were sacrificed 2 weeks after the last immunization and splenocytes isolated from them were stimulated with irradiated HEVc-Flk cells for 5 days. Thereafter, cytotoxicity assays were performed with parental HEVc (a) or HEVc-Flk (▪) as target cells. (B) Effector cells isolated from control group of mice immunized only with empty vector. (C) Effector cells from the pKd vaccinated group of mice. (D) Effector cells from the positive control group immunized with the Flk-1 whole gene vaccine.
FIG. 16. (A) Masson's trichrome staining of CT-26 lung metastasis tumor sections prepared 19 days after tumor cell challenge. The blue arrows indicate the blood vessels in the tumor. (B) Matrigel assay. Matrigel was implanted into mice vaccinated with either empty vector or pKd vaccines. Quantification of vessel growth by fluorimetry after staining of endothelium with FITC-labeled isolectin B4. The average fluorescence of Matrigel plugs from each group of mice is depicted by the bar graph (n=4; mean±SD). *, P<0.05 compared to empty vector control group.
FIG. 17 illustrates minigenes encoding the murine pHI-Dd and pHI-Kb, which comprise immunogenic Flk-1 peptide fragments separated by AAA spacers, and including a HIVtat peptide at the N-terminus thereof. Vectors were assembled by PCR with overlapping oligonucleotides as templates as shown in the figure.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
A DNA composition effective for inhibiting endothelial cell proliferation comprises a DNA construct that encodes a vascular endothelial growth factor (VEGF) receptor or at least one immunogenic fragment thereof capable of eliciting an immune response against vascular endothelial cells, incorporated in a pharmaceutically acceptable carrier. The VEGF receptor or immunogenic fragment thereof is expressible in immune cells. The term “DNA construct” as used herein and in the appended claims means a synthetic DNA structure that can be transcribed in target cells. The construct can comprise a linear nucleic acid, such as a purified DNA, or preferably, DNA incorporated in a plasmid vector. The DNA can also be incorporated in a viral or bacterial vector, preferably an attenuated viral or bacterial vector that is non-pathogenic. The DNA construct is capable of being expressed in immune cells (e.g., macrophages and/or dendritic cells) in a subject to which the DNA composition has been administered. As used herein and in the appended claims, the term “VEGF receptor polypeptide” includes full length VEGF receptors, as well as immunogenic fragments thereof, as described herein.
Suitable DNA constructs are those that encode a VEGF receptor such as VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6), e.g., DNA sequences SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO 5, respectively, or an immunogenic fragment (i.e., an epitope) thereof. Preferably, the DNA construct encodes two or more immunogenic VEGF receptor fragments linked together in linear fashion by a linker peptide between each fragment.
Five VEGF sub-types have been identified, including VEGF-1 (also known as VEGF-A), VEGF-2 (also known as VEGF-C), VEGF-B, VEGF-D and VEGF-E. See, for example, U.S. Pat. No. 6,235,713 to Achen et al. and references cited therein. VEGF receptors are protein-tyrosine kinases specific to endothelial cells. Several receptor protein-tyrosine kinases that are specific to endothelial cells have been identified, including Flt-1 (VEGF receptor 1; VEGFR-1), KDR (VEGFR-2), Flk-1 (the murine homolog of KDR), Flt-4 (VEGFR-3), Tie, Tie-2 and Tek, several of which are VEGF receptors.
Preferably, the compositions of the present invention comprise a DNA construct that encodes one or more VEGF receptor protein, such as a tyrosine kinase that is specific to endothelial cells, including, for example Flt-1, KDR, Flk-1, functional homologs thereof, or at least one immunogenic fragment (epitope) thereof. The functional homologs preferably share at least about 80% homology with the aforementioned VEGF receptor proteins. Immunogenic fragments of VEGF receptors preferably consist of about 8 to 10 contiguous amino acid resides from a VEGF receptor, i.e., from an epitope region of the protein.
In some preferred embodiments, the DNA construct encodes two or more (e.g., 2 to 5) immunogenic fragments of a VEGF receptor. The DNA construct preferably encodes the fragments linked together by a spacer peptide (e.g., AAA or AAY) between each fragment. Preferably, the DNA construct encoding an immunogenic fragment of a VEGF receptor also encodes a membrane-translocating peptide, such as the HIVtat peptide, connected to the N-terminus of the immunogenic fragment of legumain by a spacer peptide. When the DNA construct encodes two or more immunogenic fragments, the membrane-translocating peptide preferably is linked to the first fragment from the N-terminus thereof.
Immunogenic fragments of VEGF receptors can be identified by the HLA Binding Predictions program provided by the Bioinformatics & Molecular Analysis Section (BIMAS) of the National Institutes of Health (NIH) at the NIH www website, which is incorporated herein by reference.
The DNA compositions of the present invention stimulate formation of CTLs that are active against proliferating endothelial cells that overexpress a VEGF receptor. Because VEGF receptors are only substantially expressed on proliferating endothelial cells, a CTL that forms in response to contact with a composition of the invention will substantially target only tissues where active angiogenesis (e.g., vascularization) is occurring. Non-proliferating endothelial cells, such as the linings of established blood vessels, are substantially lacking in VEGF receptor antigens and are thus not affected by a CTL elicited by the DNA composition of the invention.
In a preferred embodiment, the DNA composition comprises a polynucleotide sequence that operably encodes at least one immunogenic fragment of a VEGF receptor incorporated in a vector capable of expressing the immunogenic fragment in an immune cell after being taken up by the immune cell. This composition can promote activation of naive T cells, both directly and indirectly, through the intervention of dendritic cells.
In some preferred embodiments, the DNA composition further comprises a DNA construct encoding an immune effector protein expressible in immune cells. As used herein and in the appended claims the phrase “immune effector protein” means a protein that is involved in regulation of an immune system pathway. Preferably, the immune effector protein is a cytokine
Cytokines are proteins and polypeptides produced by cells that can affect the behavior of other cells, such as cell proliferation, cell differentiation, regulation of immune responses, hematopoiesis, and inflammatory responses. Cytokines have been classified into a number of families, including chemokines, hematopoietins, immunoglobulins, tumor necrosis factors, and a variety of unassigned molecules. See generally Oxford Dictionary of Biochemistry and Molecular Biology, Revised Edition, Oxford University Press, 2000; and C. A. Janeway, P. Travers, M. Walport and M. Schlomchik, Immunobiology, Fifth Edition, Garland Publishing, 2001 (hereinafter “Janeway and Travers”). A concise classification of cytokines is presented in Janeway and Travers, Appendix III, pages 677-679, the relevant disclosures of which are incorporated herein by reference.
Hematopoietins include, for example erythropoietin, interleukin-2 (IL-2, a 133 amino acid protein produced by T cells and involved in T cell proliferation), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-15 (a 114 amino acid IL-2-like protein, which stimulates the growth of intestinal epithelium, T cells, and NK cells), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), oncostatin M (OSM), and leukemia inhibitory factor (LIF).
Interferons include, for example, IFN-α, IFN-β, and IFN-γ (a 143 amino acid homodimeric protein produced by T cells and NK cells, which is involved in macrophage activation, increased expression of MHC molecules and antigen processing components, IG class switching, and suppression of TH2).
Immunoglobulins include, for example, B7.1 (CD80), and B7.2 (CD86), both of which co-stimulate T cell responses.
The tumor necrosis factor (TNF) family includes, for example, TNF-α, TNF-β (lymphotoxin), lymphotoxin-β (LT-β), CD40 ligands, Fas ligand, CD27 ligand, CD30 ligand, 4-1BB ligand, Trail, and OPG ligand.
The biological roles of CD40 ligand (CD40L), particularly its interaction with CD40 expressed on antigen presenting cells during costimulation of T cell activation, are well known in the art. CD40 is a 48 kDa glycoprotein expressed on the surface of all mature B cells, most mature B-cell malignancies, and some early B-cell acute lymphocytic leukemias, but it is not expressed on plasma cells, Clark, Tissue Antigens 1990, 35:33-36. CD40L, a type II membrane protein of about 35 kDa, is expressed on the surface of T cells upon antigen recognition. Members of the TNF family are biologically most active when expressed as homotrimers. CD40L is no exception in this regard and can be expressed as a homotrimer (CD40LT) by modification of a 33 amino acid leucine zipper motif fused to the N-terminus of the entire extracellular domain of this ligand. CD40LT DNA has been reported by Gurunathan et al. J. Immunol. 1998, 161:4563, to enhance cellular immune responses such as induction of IFN-γ and cytolytic T cell activity when mice were vaccinated with DNA encoding the highly immunogenic model antigen β-galactosidase.
CD40LT is an important factor in the activation of T cells necessary to induce an effective protective immunity against tumor self-antigens. Once MHC class I antigen:peptide complexes are taken up by dendritic cells (DCs) and presented to naive T cells, the first antigen signal is delivered via T cell receptors (TCR), followed by upregulation of CD40LT. On the T cell surface, CD40LT then induces costimulatory activity on DCs via CD40-CD40LT interactions. Thus primed, these APCs can express costimulatory molecules B7.1 (CD80) and B7.2 (CD86), which sends a second costimulatory signal to T cells via interaction with CD28, an event required for full activation of T cells to concurrently produce pro-inflammatory cytokines INF-γ and IL12, and to perform effector functions.
Various cytokines that are not assigned to a particular family include, for example, tumor growth factor-β (TGF-β), IL-1α, IL-1β, IL-1 RA, IL-10, IL-12 (natural killer cell stimulatory factor; a heterodimer having a 197 amino acid chain and a 306 amino acid chain, which is involved in NK cell activation and induction of T cell differentiation to TH1-like cells), macrophage inhibitory factor (MIF), IL-16, IL-17 (a cytokine production-inducing factor, which induces cytokine production in epithelia, endothelia, and fibroblasts), and IL-18.
Chemokines are a family of cytokines that are relatively small chemoattractant proteins and polypeptides, which stimulate the migration and activation of various cells, such as leucocyte migration (e.g., phagocytes and lymphocytes). Chemokines play a role in inflammation and other immune responses. Chemokines have been classified into a number of families, including the C chemokines, CC chemokines, CXC chemokines, and CX3C chemokines. The names refer to the number and spacing of cysteine (c) residues in the molecules; C chemokines having one cysteine, CC chemokines having two contiguous cysteines, CXC having two cysteines separated by a single amino acid residue, and CX3C chemokines having two cysteines separated by three amino acid residues. Chemokines interact with a number of chemokine receptors present on cell surfaces. See Janeway and Travers, Appendix IV, page 680, which is incorporated herein by reference.
In addition, chemokines can have immunomodulating activity and have been implicated in immune responses to cancer. For example, murine 6Ckine/SLC, the mouse analog of the human secondary lymphoid tissue chemokine (SLC), now commonly referred to as CCL21, has been reported to induce an antitumor response in a C-26 colon carcinoma tumor cell line. See Vicari, et al. J. Immunol. 2000; 165(4):1992-2000. Human CCL21 and its murine counterpart, 6Ckine/SLC, are classified as CC chemokines, which interact with the CCR7 chemokine receptor. Murine 6Ckine/SLC (muCCL21) is also reported by Vicari et al. to be a ligand for the CXCR3 chemokine receptor. Human CCL21, murine muCCL21 and a variety of other chemokines are implicated in the regulation of various immune system cells such as dendritic cells, T cells, and natural killer (NK) cells.
Mig and IP-10 are CXC chemokines that interact with the CXCR3 receptor, which is associated with activated T cells. Lymphotactin is a C chemokine, which interacts with the XCR1 receptor associated with T cells and NK cells. Fractalkine is a CX3C chemokine, which interact with the CX3CR1 receptor that is associated with T cells, monocytes and neutrophils.
Particularly preferred immune effector proteins to be encoded by the DNA compositions of the present invention include cytokines IL-2 (a hematopoietin), CCL21 (a chemokine), as well as CD40 ligands such as CD40 ligand trimer (CD40LT), a TNF family cytokine.
DNA and protein sequences for human IL-2 have been published in GenBank, Accession No. BC070338, the disclosures of which are incorporated herein by reference. The DNA and protein sequences of murine IL-2 have been in GenBank, Accession No. NM 008366, the disclosures of which are incorporated herein by reference.
DNA and protein sequences for human CCL21 have been published in GenBank, Accession No. AB002409, the disclosures of which are incorporated herein by reference.
Human CD40 ligand (CD40L) is a 261 amino acid protein, which exists as a trimer (CD40LT) in its most active form. The DNA sequence encoding human CD40L (also known as CD154) has been published in GenBank, Accession No. NM 000074, the disclosure of which is incorporated herein by reference.
A DNA composition of the invention can be utilized to provide long term inhibition of tumor growth and/or tumor metastases in a patient treated with the composition. In a preferred embodiment, the DNA composition is administered in conjunction with an antitumor chemotherapeutic agent. The DNA composition can be administered together with the chemotherapeutic agent in a combined dosage form, or the composition and chemotherapeutic agent can be administered in separate dosage forms and at separate dosage intervals tailored to the pharmacology of the chemotherapeutic agent being administered.
Chemotherapeutic agents useful in combination with the DNA compositions of the present invention include antitumor agents such as doxorubicin, paclitaxol, a cyclophosphamide, etoposide, 5-fluorouracil, methotrexate, and the like.
As used herein, the term “immunity” refers to long term immunological protection against the virulent form of the infectious agent or tumor antigen. The term “immunization” refers to prophylactic exposure to an antigen of a pathogenic agent derived from a non-virulent source, which results in immunity to the pathogen in the treated subject.
A DNA composition of the present invention preferably comprises a nucleotide sequence that encodes a VEGF receptor or at least an immunogenic fragment of a VEGF receptor protein, operably linked to regulatory elements needed for gene expression in immune cells of the subject to be treated.
Useful DNA constructs preferably include regulatory elements necessary for expression of nucleotides. Such elements include, for example, a promoter, an initiation codon, a stop codon, and a polyadenylation signal. In addition, enhancers are often required for expression of a sequence that encodes an immunogenic target protein. As is known in the art, these elements are preferably operably linked to the sequence that encodes the desired polypeptide. Regulatory elements are preferably selected that are operable in immune cells of the subject to which they are to be administered.
Initiation codons and stop codons are preferably included as part of a nucleotide sequence that encodes the immunogenic fragment of the VEGF receptor protein in a DNA composition of the present invention. The initiation and termination codons must be in frame with the coding sequence.
Promoters and polyadenylation signals included in a composition of the present invention are preferably selected to be functional within the cells of the subject to be treated.
Examples of promoters useful in the compositions of the present invention, especially in the production of a composition for use in humans, include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human actin, human myosin, human hemoglobin, human muscle creatine, and human metalothionein.
Examples of polyadenylation signals useful in the compositions of the present invention, especially in the production of a composition for humans, include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals.
In addition to the regulatory elements required for DNA expression, other elements can also be included in the DNA molecule. Such additional elements include enhancers. The enhancer can be, for example, human actin, human myosin, human hemoglobin, human muscle creatine and viral enhancers such as those from CMV, RSV and EBV.
Regulatory sequences and codons are generally species dependent, so in order to maximize protein production, the regulatory sequences and codons are preferably selected to be effective in the species to be immunized. One having ordinary skill in the art can produce DNA constructs that are functional in a given subject species.
The DNA constructs of the present compositions can be “naked” DNA as defined in Restifo et al. Gene Therapy 7, 89-92 (2000), the pertinent disclosure of which is incorporated by reference. Alternatively, the DNA can be operably incorporated in a vector. Useful delivery vectors include biodegradable microcapsules, immuno-stimulating complexes (ISCOMs) or liposomes, and genetically engineered attenuated live vectors such as viruses or bacteria.
Examples of suitable attenuated live bacterial vectors include Salmonella typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus, Bacille Calmette-Guerin (BCG), Escherichia coli, Vibrio cholerae, Campylobacter, or any other suitable bacterial vector, as is known in the art. Methods of transforming live bacterial vectors with an exogenous DNA construct are well described in the art. See, for example, Joseph Sambrook and David W. Russell, Molecular Cloning, A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001).
A preferred attenuated bacterial vector is an attenuated Salmonella carrier, which is the doubly attenuated strain of S. typhimurium designated as RE 88, and which includes the dam− and AroA− mutations, available from Remedyne Corporation (Goleta, Calif.). The attenuated Salmonella carrier is engineered to include DNA encoding a VEGF receptor polypeptide such as an immunogenic fragment of a VEGF receptor, which is expressible in immune cells of a mammal to which it is administered. The bacteria do not themselves express the immunogenic polypeptide, but rather deliver DNA encoding the polypeptide to immune cells, such as macrophages and dendritic cells (DCs), which in turn express the polypeptide. Such compositions can provide prolonged antitumor effects in murine models. Furthermore, in vivo depletion experiments of T cells indicated the involvement of CD8+ but not CD4+ T cells. The cytotoxic effect mediated by CD8+ T cells in vitro is specifically directed against target proliferating endothelial cells that overexpress the VEGF receptors.
Preferred viral vectors include Bacteriophages, Herpes virus, Adenovirus, Polio virus, Vaccinia virus, and Avipox. Methods of transforming viral vector with an exogenous DNA construct are also well described in the art. See Sambrook and Russell, above.
Liposome vectors are unilamellar or multilamellar vesicles, having a membrane portion formed of lipophilic material and an interior aqueous portion. The aqueous portion is used in the present invention to contain the polynucleotide material to be delivered to the target cell. It is generally preferred that the liposome forming materials have a cationic group, such as a quaternary ammonium group, and one or more lipophilic groups, such as saturated or unsaturated alkyl groups having about 6 to about 30 carbon atoms. One group of suitable materials is described in European Patent Publication No. 0187702, and further discussed in U.S. Pat. No. 6,228,844 to Wolff et al., the pertinent disclosures of which are incorporated by reference. Many other suitable liposome-forming cationic lipid compounds are described in the literature. See, e.g., L. Stamatatos, et al., Biochemistry 27:3917-3925 (1988); and H. Eibl, et al., Biophysical Chemistry 10:261-271 (1979). Alternatively, a microsphere such as a polylactide-coglycolide biodegradable microsphere can be utilized. A nucleic acid construct is encapsulated or otherwise complexed with the liposome or microsphere for delivery of the nucleic acid to a tissue, as is known in the art.
The method aspects of the present invention comprise the step of administering DNA compositions of the invention to a mammal, such as a human. In some preferred embodiments, the DNA polynucleotides are administered orally, intramuscularly, intranasally, intraperitoneally, subcutaneously, intradermally, or topically.
In a method aspect of the present invention, a DNA composition of the invention can be utilized as a vaccine to provide long term inhibition of endothelial cell proliferation in a patient treated with the vaccine. In one preferred method embodiment, a DNA composition comprising a polynucleotide construct operably encoding a VEGF receptor polypeptide is administered to a mammal in need of inhibition of endothelial cell proliferation, in an amount that is sufficient to elicit an immune response against proliferating endothelial cells. The terms “DNA composition” and “DNA vaccine” are used interchangeably herein for convenience, with respect to the methods of the present invention.
The present invention also provides a method of inhibiting angiogenesis in a mammal treated with the DNA vaccine. In such a method embodiment, a vaccine comprising a DNA construct operably encoding a VEGF receptor polypeptide is administered to a mammal suffering from an angiogenesis related disease, in an amount sufficient to elicit an immune response against proliferating endothelial cells.
In yet another method aspect of the present invention, tumor growth is inhibited by treatment of a mammal with a DNA vaccine. In such a method embodiment, an immune response eliciting amount of a vaccine comprising a DNA construct operably encoding a VEGF receptor polypeptide is administered to a mammal having a growing tumor. Treatment with the vaccine results in tumor growth arrest by immunizing the mammal against proliferating endothelial cells. Destruction of proliferating endothelial cells by the mammal's immune system prevents, or at least minimizes vascularization of the tumor.
In the method embodiments of the present invention, the vaccines can be administered enterally, such as by oral administration, or by intramuscular injection. Preferably, the mammal treated with the inventive vaccine is a human. A patient suffering from cancer, such as lung or colon carcinoma, or prostate tumors, diabetic retinopathy, and the like, can benefit from immunization by the vaccines of the present invention.
Compositions of the present invention preferably are formulated with pharmaceutically acceptable carriers or excipients such as water, saline, dextrose, glycerol, and the like, and combinations thereof. The compositions can also contain auxiliary substances such as wetting agents, emulsifying agents, buffers, and the like.
The compositions of the present invention are preferably administered orally to a mammal, such as a human, as a solution or suspension in a pharmaceutically acceptable carrier, at a DNA concentration in the range of about 1 to about 10 micrograms per milliliter. The appropriate dosage will depend upon the subject to be vaccinated, and in part upon the judgment of the medical practitioner administering or requesting administration of the vaccine.
The compositions of the present invention can be packaged in suitably sterilized containers such as ampules, bottles, or vials, either in multi-dose or in unit dosage forms. The containers are preferably hermetically sealed after being filled with a compositions of the invention. Preferably, the compositions are packaged in a container having a label affixed thereto, which label identifies the compositions, and bears a notice in a form prescribed by a government agency such as the United States Food and Drug Administration reflecting approval of the vaccine under appropriate laws, dosage information, and the like. The label preferably contains information about the composition that is useful to an health care professional administering the compositions to a patient. The package also preferably contains printed informational materials relating to the administration of the composition, instructions, indications, and any necessary required warnings.
The amino acid sequences of VEGF receptor proteins have been disclosed in the art, as have the nucleic acid sequences encoding these proteins. The nucleic acid sequence encoding KDR (FIG. 1, SEQ ID NO: 1), and its corresponding protein sequence (FIG. 2, SEQ ID NO: 2) have been published by Yu et al., in the EMBL database of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK (EMBL accession number is EMBL:AF063658), the disclosures of which are incorporated herein by reference. The nucleic acid sequence encoding Flt-1 (FIG. 3, SEQ ID NO: 3), and its corresponding protein sequence (FIG. 4, SEQ ID NO: 4) have been published by Yu et al., in the EMBL database of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK (EMBL accession number is EMBL:AF063657), the disclosures of which are incorporated herein by reference.
The nucleic acid sequence encoding Flk-1, and its corresponding protein sequence have been published by Mathews et al., Proc. Natl. Acad. Sci. USA 1991, 88:9026-9030, and the structures were corrected by Quinn et al., Proc. Natl. Acad. Sci. USA 1991, 90:7533-7537, the relevant disclosures of which are incorporated herein by reference. The corrected DNA sequence of Flk-1 is provided in FIG. 5 as SEQ ID NO: 5, and the corrected protein sequence of Flk-1 is provided in FIG. 6 as SEQ ID NO:6.
Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence to VEGF receptor proteins such as KDR, Flk-1 and Flt-1, can be used in the practice of the invention. Such DNA sequences include those which are capable of hybridizing to the VEGF receptor sequences as well. Preferably the functionally equivalent homologs of the VEGF receptor protein DNA shares at least about 80% homology with the DNA encoding the aforementioned VEGF receptor proteins.
Altered DNA sequences which can be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product. The gene product itself may contain deletions, additions or substitutions of amino acid residues within the VEGF receptor sequences, which result in a silent change, thus producing a functionally equivalent VEGF receptor proteins. Such amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine. As used herein, a functionally equivalent VEGF receptor refers to a receptor that binds to VEGF or fragments thereof, but not necessarily with the same binding affinity of its counterpart native KDR, Flk-1 or Flt-1.
The DNA constructs may be engineered in order to alter the VEGF receptor coding sequence for a variety of ends including, but not limited to, alterations that modify processing and expression of the gene product. For example, mutations may be introduced using techniques that are well known in the art, e.g. site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, phosphorylation, etc.
Mouse Flk-1 (SEQ ID NO: 6) shares an approximately 85% homology with human KDR (SEQ ID NO: 2) and plays an analogous role in mouse physiology to the role of KDR in humans. In fact, VEGFR-2 is often referred to as KDR/Flk-1, reflecting the close analogy between these two VEGF receptor homologs. For this reason, treatment of mice with a DNA vaccine of the invention, encoding Flk-1 (e.g., DNA SEQ ID NO: 5) was chosen as a suitable model for human DNA vaccines encoding KDR.
The following examples are provided to further illustrate the features and embodiments of the present invention, and are not meant to be limiting.
MATERIALS, METHODS AND EXAMPLES
Materials. C57/BL/6J and Balb/C mice were obtained from the Scripps Research Institute breeding facility. The murine tumor cell lines used for evaluation included the melanoma cell line B16 and the colon carcinoma cell line CT26, all of which were obtained from Dr. I. J. Fidler, MD Anderson Cancer Center, Houston, Tex. The murine Lewis lung cancer cell line D121 was obtained from Dr. Lea Eisenbach, Weizmann Institute, Rehovot, Israel. The DNA encoding Flk-1 was kindly provided by Dr. Lemischka (Princeton University, Princeton, N.J.), and was cloned into the pcDNA3.1 eucaryotic expression vector provided by Invitrogen, Huntsville, Ala., using the restriction sites KpnI and XbaI. An attenuated strain of Salmonella typhimurium was provided by B. A. D. Stocker (Stanford University, Stanford, Calif.). Antibodies were obtained from BD Biosciences, Bedford, Mass. T-STIM culture supplement was obtained from BD Biosciences, Bedford, Mass. Fluorescein isothiocyanate (FITC) and R-Phycoerythrin (PE) were obtained from Molecular Probes, Eugene, Oreg. FITC-labeled and PE-labeled antibodies were prepared according to the manufacturer's recommended protocols.
EXAMPLE 1
Preparation of a DNA Vaccine Encoding Flk-1
The pcDNA3.1 vector containing Flk-1 DNA (SEQ ID NO: 5; about 10 pg to about 0.1 μg of pDNA) was electroporated into freshly prepared attenuated Salmonella typhimurium, utilizing a Bio-Rad Pulser at 2.5 kV, 25 μF, and 200 Ohm according to the manufacturer's recommended procedures. Salmonella containing the vector were selected on ampicillin-containing plates. Colonies were picked the next day and cultured overnight in LB broth (EM Science, Gibbstown, N.J.) with ampicillin added. The bacteria were isolated and washed in phosphate buffered saline (PBS). The washed bacteria were then suspended in PBS medium at a concentration of about 1×109 recombinant Salmonella per milliliter of PBS, to form a vaccine solution for later use. The vaccine was stored in sealed ampules until used. A “control vaccine” consisting of Salmonella transformed with the pcDNA3.1 vector alone (no Flk-1 DNA) was also prepared according to the same procedure. The plasmid DNA was stored at about −80° C. before transforming the Salmonella. 
EXAMPLE 2
Vaccination of Mice with a DNA Vaccine Encoding Flk-1
Balb/C mice (about 6 mice per treatment group) were vaccinated with the DNA vaccine of Example 1 (about 1×108 recombinant Salmonella in about 100 μl of PBS) by oral gavage, three times at two week intervals. Another group of mice were vaccinated with control vaccine (consisting of attenuated Salmonella containing the empty vector) according to the same schedule as the mice vaccinated with the inventive vaccine.
EXAMPLE 3
Evaluation of Tumor Resistance of Vaccinated Mice
About two weeks after the third vaccination, Balb/C mice from Example 2 (about 6 mice per treatment group) were challenged with either about 1×105 B16 melanoma cells (subcutaneously), about 1×105D121 Lewis lung carcinoma cells (subcutaneously), or about 7.5×104 CT26 colon carcinoma cells (intravenously). The subcutaneous Lewis lung tumors were surgically removed after about two weeks of growth to allow spontaneous dissemination to the lung. Subcutaneous tumor growth was measured in two dimensions every other day, and tumor volume was calculated according to the formula:

volume=(width2)(length÷2)

for each tumor. The amount of spontaneous metastasis of D121 to the lungs was evaluated about 30 days after removal of the subcutaneous primary tumor. The mice were sacrificed and necropsied, and the tumor burdens of the lungs were evaluated according to the percentage of the lung surface that was covered by tumor and scored as “0” for no tumor, “1” for less than about 20% tumor coverage, “2” for about 20 to about 30% tumor coverage, and “3” for greater than about 50% tumor coverage. FIG. 7 shows pictures of lungs from three mice challenged with D121 Lewis lung carcinoma cells. The lower lung was scored 1, whereas the upper two lungs were scored 3, having a large proportion of the lung surface covered by tumors. Animals that died prior to the 30 day evaluation were given a “+” score.

The results of these evaluations are provided in Tables 1-4, and in FIGS. 8-10, discussed in detail below.







TABLE 1







Tumor Metastasis in Balb/C Mice Challenged with


D121 Lewis Lung Carcinoma Cells.








Mouse Vaccination Group
Metastatic Scores





Control - vaccination with untransformed Salmonella
3,3,3,3,+,+


Control - vaccination with control vaccine
3,3,3,3,+,+


(containing empty vector)



Vaccination with DNA Vaccine of Example 1
0,0,1,1,1,2,2


(containing Flk-1)









The Balb/C mice that were challenged by intravenous injection of CT-26 colon carcinoma cells were evaluated for mortality over about a 63 day (7 week) period. Mortality information is presented in Table 2 below, and graphically illustrated in FIG. 8.
In FIG. 8, the % survival of mice treated with the inventive vaccine of Example 1 is indicated by the heavy, solid line at 100% survival. The % survival of naive mice (no vaccination) challenged with the C26 cell is indicated by the solid, thin line, whereas, the % survival of the mice treated with the control vaccine (no Flk-1 DNA) is indicated by the dot-dash line.







TABLE 2







Suppression of Mortality in Balb/C Mice Immunized With the


Vaccine of Example 1 and Challenged with CT 26 Carcinoma.











% Survival
% Survival 
% Survival


Treatment
on Day 30
on Day 36
on Day 63













Control, No Vaccine
50
0
0


Control Vaccine
33
0
0


Vaccine of Ex. 1
100
100
100









The suppression of growth of the primary (subcutaneous) tumor in D121 challenged Balb/C mice was evaluated by determination of primary tumor volume at day 14 after challenge. Results are presented in Table 3 below, and graphically illustrated in FIG. 9.
In FIG. 9, the first bar, labeled “PBS” indicates mice that were not vaccinated (naive mice), the middle bar, labeled “empty vector” indicates mice treated with the control vaccine, and the third bar, labeled “pcDNA3.1-Flk1” indicates mice immunized with the inventive vaccine of Example 1.







TABLE 3







Suppression of Subcutaneous D121 Carcinoma Tumor in


Balb/C Mice Immunized With the Vaccine of Example 1._












Tumor volume
Standard



Treatment
mm3
Deviation















Control No Vaccine
665
227



Control Vaccine
641
157



Vaccine of Ex. 1
183
35










Suppression of subcutaneous B16 melanoma tumor growth was evaluated by monitoring the subcutaneous tumor volume over a period of about 17 days after tumor challenge. Results are presented in Table 4 and graphically illustrated in FIG. 10 below. In FIG. 10, average tumor volume data indicated by (●) represents mice immunized with the inventive vaccine of Example 1, whereas data indicated by (◯) indicates mice treated with the control vaccine.







TABLE 4







Suppression of Subcutaneous B16 Melanoma Tumor in


Balb/C Mice Immunized With the Vaccine of Example 1.









Tumor Volume (mm3) on Day











Treatment
0
9
14
17





Control Vaccine
0
907
1273 
4213


Vaccine of Ex. 1
0
447
462
1063


% Tumor Suppression
—
    51%
    64%
    75%









EXAMPLE 4
Upregulation of CD25, CD69 and CD2 Activation Markers in Splenocytes (CD8+ T Cells) from Vaccinated Mice
C5/7BL/6J mice (about 4 mice per treatment group) were vaccinated with the DNA vaccine of Example 1 and the control vaccine (no Flk-1) as described in Example 2. Splenocytes were isolated from the immunized mice and the control mouse group about six weeks after the third vaccination. The splenocyte cells were cultured for 24 hours together with cells from a B16 melanoma cell line transduced to express Flk-1 and with untransformed B16 cells in T cell medium (about 5 mL per culture) containing about 4% by volume of T-STIM culture supplement (Cat. #354115, BD Biosciences, Bedford, Mass.). The cells were then stained with FITC-conjugated CD8+ antibody and PE-conjugated antibodies of CD25, CD69, and CD2. The cell suspensions were evaluated using a Becton Dickenson FAC scan to determine the percentage of CD8+ T cells positive for CD25 and CD69 for each splenocyte/B16 melanoma cell combination. The results are presented in Table 5 and are illustrated graphically in FIG. 11.







TABLE 5







Upregulation of CD25, CD69 and CD2 Activation


Markers in Splenocytes From Vaccinated Mice













% CD25
% CD69
CD2 positive



Treatment
positive
positive
mean fluorescence
















Control vaccine +
9
18
570 mfu



B16-Flk-1 cells






DNA vaccine + 
12
29
550 mfu



B16 cells






DNA vaccine +
21
35
700 mfu



B16-Flk-1 cells










mfu = mean fluorescence units.






The results provided in Tables 1-5 and FIGS. 8-11 demonstrate that the DNA vaccine of Example 1, comprising a DNA encoding Flk-1, the murine analog of KDR, can effectively immunize mice against a variety of tumor forming cancer cells. Although not intending to be bound by theory, it is believed that the vaccine acts by inhibiting angiogenesis in the tumor, i.e., preventing new blood vessel formation and effectively starving the tumor.
The data in Table 1 demonstrate that the inventive vaccine of Example 1 leads to a suppression of tumor metastasis to the lungs of mice challenged with D121 Lewis lung carcinoma. None of the mice immunized with the vaccine of Example 1 died, and all had less than about 50% tumor coverage on the lungs (2 had <20%). In contrast, two mice died from each control group and all of the remaining mice had greater than about 50% tumor coverage on the lungs.
The inventive vaccine of Example 1 also significantly decreased mortality of Balb-C mice that were challenged intravenously by CT-26 colon carcinoma cells, as demonstrated by the data in Table 2 and FIG. 8. All of the mice immunized with the vaccine of Example 1 survived the entire 63 day observation period after challenge. In the control groups, however, all of the mice had died by day 36 post challenge.
As demonstrated by the data in Table 3 and FIG. 9, subcutaneous D121 Lewis lung carcinoma tumor growth was suppressed by immunization with the inventive vaccine of Example 1 by a factor of about 4.3 to about 4.5, relative to the control mouse groups treated with no vaccine or the control vaccine.
Similarly, as shown in Table 4 and FIG. 10, subcutaneous B16 melanoma tumor growth was suppressed by a factor of about 4 in mice immunized with the inventive vaccine of Example 1, relative to tumor growth in the control group.
The data in Table 5 and FIG. 11 show that splenocytes isolated from C57/BL/6J mice vaccinated with the DNA vaccine of Example 1 exhibited an upregulation of CD2, CD25 and CD69 activation markers relative to the control group of mice, when cultured with B16 melanoma cells transformed to present Flk-1 antigen.
The following additional non-limiting Examples further illustrate the compositions and methods of the present invention in which the VEGF receptor polypeptide is an immunogenic fragment of a VEGF receptor.
EXAMPLE 5
DNA Composition Encoding Immunogenic Fragments of a VEGF Receptor and Evaluation Therefor in a BALB/c Mouse Model
Animals, Bacterial strains, and cell lines: Female BALB/c mice were purchased from the Jackson Laboratory. All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The murine D2F2 breast cancer cell line was kindly provided by Dr. W-Z. Wei (Karmanos Cancer Institute, Detroit, Mich., USA). The murine colon carcinoma cell line CT-26 was provided by Dr. I. J. Fidler (MD Anderson Cancer Center, Houston, Tex.). The murine high endothelial venule cell line (HEVc) was a gift from Dr. J. M. Cook-Mills (University of Cincinnati, Ohio, USA). The HEVc-Flk-1 cell line was established by retroviral transduction with the Flk-1 gene by Drs. Harald Wodrich and Andreas G. Niethammer. The doubly attenuated S. typhimurium (AroA-, dam-) strain RE88 was kindly provided by Remedyne Corporation (Santa Barbara, Calif.) and was transduced with DNA vaccine plasmids to serve as a vaccine carrier, as described above.
Construction of Expression Vectors. Vector construction is illustrated schematically by FIG. 12, Panel A. The expression vectors were established based on the pcDNA/Myc/H is vector (Invitrogen, Carlsbad, Calif.) containing the ubiquitin sequence. The peptides were cloned downstream of ubiquitin, and the sequence of each peptide is indicated in Table 6. All peptides were engineered to be in-frame with the myc epitope. Constructs were confirmed by DNA sequencing at The Scripps Research Institute's Core Facility (La Jolla, Calif.). Peptide expression was demonstrated by Western blotting analysis of transfected 293 T cells with monoclonal anti-myc antibody (Invitrogen). Once peptide expression was verified, a stop codon was introduced immediately in front of the myc epitope sequences. The resulting vectors, pDd, pKd, pDd+Kd1, and pKd+Dd2 were each verified by nucleotide sequencing and used to transform doubly attenuated S. typhimurium (dam-, AroA-) for subsequent immunization.







TABLE 6







Immunogenic Flk Fragments.












Peptide 

SEQ ID 
Included in



Name
Sequence
NO:
vaccine
















Flk54
RGQRDLDWL
7
pDd; pDd + Kd1



 



Flk210
TYQSIMYIV
8
pKd; pKd + Dd2



 



Flk221
VGYRIYDVI
9
pDd; pKd + Dd2



 



Flk366
WYRNGRPIE
10
pKd; pDd + Kd1



 



Flk438
QYGTMQTLT
11
pKd: pKd + Dd2



 



Flk741
LGCARAETL
12
pDd; pDd + Kd1



 



Flk792
EGELKTGYL
13
pDd; pKd + Dd2



 



Flk993
LYKDFLYTE
14
pKd; pDd + Kd1



 



Flk1147
QRPSFSELV
15
pDd; pDd + Kd1



 



Flk1153
ELVEHLGNL
16
pKd; pKd + Dd2










Oral Immunization and Tumor Cell Challenge. Groups of BALB/c mice were immunized 3 times at 1-week intervals by gavage with 100 μl PBS containing approximately 5×108 CFU of RE88 S. typhimurium harboring either empty vector, pDd, pKd, pDd+Kd1 and pKd+Dd2 plasmids. Mice were challenged i.v. with different carcinoma cell lines 2 weeks after the last immunization.
Cytotoxicity Assay. Cytotoxicity was measured by a standard 51Cr-release assay as previously described (Zhou, H. et al. J. Clin. Invest., 2004; 113: 1792-1798). The percentage of specific target cell lysis was calculated by the formula [(E−S)/(T−S)]×100, where E is the average experimental release, S is the average spontaneous release, and T is the average total release.
Evaluation of anti-angiogenic activity. Suppression of angiogenesis was determined by the Matrigel assay as previously described (Niethammer et al. Nat. Med., 2002, 8: 1369-1375). Vessel growth in the Matrigel was determined with fluorimetry by staining the endothelium using FITC-labeled isolectin B4.
Statistical Analysis. The statistical significance of differential findings between experimental groups and controls was determined by Student's t test. Findings were regarded as significant, if two-tailed P values were <0.05.
Results.
Minigenes encoded by expression vectors are expressed in mammalian cells. As shown in the previous examples, a DNA composition encoding the entire murine Flk-1 gene effectively induced CD8 T cell-mediated anti-angiogenesis that protected mice from tumor cell challenges. This example demonstrates that DNA compositions encoding an immunogenic fragment of a VEGF receptor also elicit an effective antitumor response. Five peptides were included in H-2 Dd- or H-2 Kd-restricted minigenes based on the binding predicted for these MHC class 1 molecules by the HLA Peptide Binding Predictions program provided by the BioInformatics & Molecular Analysis Section (BIMAS) of NIH, website. The amino acid sequences of these peptides are listed in Table 6.
Expression vectors were constructed based on the backbone of pcDNA/myc/His (FIG. 12, Panel A) that was modified to encode ubiquitin. Ubiquitin is useful for effective antigen processing in the proteasome leading to efficacious minigene vaccines. Gene expression of the resulting plasmids was verified by Western blotting analysis of 293 T cells transfected with either pDd-myc, pDb+Kd1-myc, pKd-myc or pKd+Dd2-myc. As expected, 293 T cells transfected with pKd-myc or pKd+Dd2-myc plasmid displayed two major bands of approximately 12 and 21 Kda, corresponding to non-ubiquitinated or ubiquitinated polypeptides, respectively (FIG. 12, Panel B). However, Western Blot analysis of 293 T cells transfected with pDd-myc and pDd+Kd1-myc showed bands of slightly higher molecular weight with the upper band being a doublet. Since these plasmids contain the correct nucleotide sequences, this phenomenon is indicative of post-translational modification, likely glycosylation. According to glycosylation prediction based on the program provided by Center for Biological Sequence Analyses at the Technical University of Denmark (DTU), website, the S1150 and S1152 amino acid residues in the Flk1147 peptide (Table 6), which were included in the pDd-myc and pDd+Kd1-myc vectors, are most likely to be glycosylated, with S1152 being almost 10 times more likely to be glycosylated than all other residues encoded by these minigenes.
The vectors pDd, pKb, pDd+Kd1 and pKd+Dd2 were generated by introducing a stop codon immediately downstream from the peptide coding sequences, so that the translated protein would not contain the myc epitope. The correct constructs were confirmed by DNA sequencing. The empty pcDNA vector was also included for control purposes, since previous data indicated that vectors containing only ubiquitin did not induce any tumor protective immune responses.
The minigene vaccines protect mice against tumors of different origin. Initially, the minigene DNA vaccines were tested in a breast carcinoma model, where mice were first vaccinated with the minigene vaccines and then challenged i.v. with murine D2F2 breast carcinoma cells (FIG. 13, upper panel). In the empty vector control group, only 25% of the mice survived 25 days after tumor cell challenge (FIG. 13). In contrast, the survival rate was 50% and 62.5% in pDd and pDd+Kd1 vaccinated groups, respectively. More importantly, the survival rate improved to 82.5% and 75% in pKd and pKd+Dd2 vaccinated groups of mice, respectively. This is quite comparable to the 75% survival rate which was observed in groups of mice immunized with the whole Flk-1 gene vaccine (FIG. 13).
To verify that the minigene vaccine designed for anti-angiogenesis is not merely specific for D2F2 breast carcinoma, its efficacy was also tested in the CT-26 murine colon carcinoma model. In this case, this pKd minigene also decreased the tumor load by 60% (FIG. 14). Similar results were also obtained by using full-length Flk-1 DNA vaccine (FIG. 14). Taken together, these data demonstrate that Flk-1 minigene vaccines also elicit protections against tumors of different origin in syngeneic BALB/c mice.
The minigene vaccines induce a CTLs response which is capable of killing Flk-1+ endothelial cells. To identify the effector cell population responsible for the vaccine induced tumor protection, in vivo depletion experiments were performed. CD8 T cells were depleted by i.p. injection of 0.5 mg/mouse 2.43 anti-CD8 monoclonal antibody 1 day before tumor cell challenge and repeated weekly (FIG. 14, upper panel). The depletion of CD8 T cells completely abrogated the pKd minigene-induced tumor protection (FIG. 14), indicating that CD8 T cells are the major effector cells.
Cytotoxicity assays were employed to demonstrate CTL activity of immunized mice. The target cells used were the murine endothelial cell line HEVc, and this same cell line stably transfected with Flk-1 plasmid (HEVc-Flk). Wild-type HEVc cells are Flk-, whereas HEVc-Flk cells readily express Flk-1 on the surface as indicated by flow cytometry analysis (FIG. 15, Panel A). Splenocytes isolated from empty vector control mice showed similar killing of HEVc-Flk cell as of HEVc parental cells (FIG. 15, Panel B). However, splenocytes from pKd-vaccinated mice induced significantly stronger killing against Flk+ target cells (FIG. 15, Panel C). This killing capacity is very similar to that of splenocytes isolated from mice immunized with the whole Flk-1 gene vaccine (FIG. 15, Panel D). These data demonstrate the specificity of the pKd minigene vaccine-induced CTL activity and its capacity to kill Flk-1+ proliferating endothelial cells.
The pKd minigene vaccine induced anti-angiogenesis effects. In an effort to investigate whether anti-angiogenesis played a key role in pKd-vaccine-induced tumor protection, tumor sections from immunized and control mice were analyzed by Masson's trichrome staining Tumor sections from empty vector control group mice showed ample, multiple blood vessels within the tumor mass. In contrast, blood vessels were rather scarce in tumor sections from pKd-vaccinated mice (FIG. 16, Panel A). These data demonstrate that immunization with the pKd vaccine resulted in the reduction of tumor vasculature.
Matrigel assays were also performed, in which blood vessel formation within the Matrigel was induced by recombinant bFGF. The difference in vessel formation was quantified by measuring the relative fluorescence intensity of extracts from Matrigel plugs obtained from immunized or control mice. Thus mice vaccinated with pKd vaccine displayed a reduction in average relative fluorescence (FIG. 16, Panel B). Taken together, these findings demonstrate that the pKd minigene vaccine induced marked anti-angiogenic effects, which aided in the protection of BALB/c mice from challenge with tumor cells of different origin in a prophylactic setting.
EXAMPLE 6
DNA Composition Encoding Immunogenic Fragments of a VEGF Receptor and Evaluation Therefor in a C57BL/6J Mouse Model
Animals, bacterial strains, and cell lines. Male or female C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, Me.). All animal experiments were performed according to the National Institutes of Health Guides for the Care and Use of Laboratory Animals, and all protocols were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.
The murine lung carcinoma cell line D121 was provided by Dr. L. Eisenbach (Weizmann Institute of Science, Rehovot, Israel). The murine prostate cancer cell line RM9 was obtained from Dr. T. C. Thompson (Baylor College of Medicine, Houston, Tex.). The murine breast cancer cell line EO771 was made available by Dr. D. Ross (University of Kentucky, Louisville, Ky.). Murine endothelial cell line, MS1, was purchased from the American Type Culture Collection (ATCC; Manassas, Va.). All cell lines were cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen, Grand Island, N.Y.), supplemented with 10% (vol/vol) fetal bovine serum (FBS).
The double-attenuated S. typhimurium (AroA−, dam−) strain RE88 was provided by the Remedyne Corporation (Santa Barbara, Calif.), and was transformed with DNA vaccine plasmids as described above.
Construction of Expression Vectors. The expression vector pCMV/ER/Myc was purchased from Invitrogen (Carlsbad, Calif.). Vector construction is illustrated schematically in FIG. 17. The following expression vectors were constructed: pHI-myc, pHI-Db-myc, pHI-Kb-myc (See FIG. 17), where the HIVtat peptide (HI) represents RKKRRQRRR (SEQ ID NO: 17). The Flk94, Flk400, and Flk1210 peptides stand for RVVGNDTGA (SEQ ID NO: 18), VILTNPISM (SEQ ID NO: 19), and FHYDNTAGI (SEQ ID NO: 20), respectively. Flk54, Flk771, and Flk1129 peptides are designated for RGQRDLDWL (SEQ ID NO: 21), VIAMFFWLL (SEQ ID NO: 22), and TTPEMHYQTM (SEQ ID NO: 23), respectively. All peptides were engineered to be in-frame with the myc epitope. Constructs were confirmed by DNA sequencing at The Scripps Research Institute's Core Facility (La Jolla, Calif.). Peptide expression was demonstrated by Western blotting with monoclonal antibody (Invitrogen, Carlsbad, Calif.). Once peptide expression was verified, a stop codon was introduced immediately in front of the myc epitope sequences. The resulting vectors, namely pHI, pHI-Db, and pHI-Kb, were verified by nucleotide sequencing and use to transform double-attenuated S. typhimurium (dam-, AroA-) for immunization. The pCMV empty vector was also included in the experiments as a control.
Peptide Synthesis. All peptides were synthesized with more than 95% purity by high-performance liquid chromatography (HPLC) by Multiple Peptide Systems (San Diego, Calif.).
Oral Immunization and Tumor-Cell Challenge. Groups of C57BL/6J mice were immunized 3 times at 1-week intervals by gavage with 100 μL phosphate-buffered saline (PBS) containing approximately 5×108 double-attenuated RE88 S. typhimurium harboring either pCMV, pHI, pHI-Db, or pHI-Kb plasmids. Mice were challenged intravenously with different carcinoma cells 2 weeks after the last immunization.
Cytotoxicity and ELISPOT assays and in vivo depletion. Cytotoxicity was measured by a standard 51Cr-release assay as described above. The percentage of specific target cell lysis was calculated by the formula [(E−S)/(T−S)]×100, where E is the average experimental release, S the average spontaneous release, and T the average total release.
Enzyme-linked immunospot (ELISPOT) assays were performed with an ELISPOT kit (PharMingen, La Jolla, Calif.) according to the instructions provided by the manufacturer.
In vivo depletion was performed on vaccinated mice by intraperitoneal injection of anti-CD4 antibody (GK1.5, 0.4 mg/mouse) or anti-CD8 antibody (2.43, at 0.6 mg/mouse) 1 day before tumor challenge and repeated weekly.
Evaluation of Antiangiogenic Effects. Two weeks after the last vaccination, mice were given subcutaneous injections in the sternal region with 400 μL growth factor-reduced Matrigel (BD Biosciences, San Jose, Calif.) containing 400 ng/mL basic fibroblast growth factor (PeproTech, Rocky Hill, N.J.). In all mice, the endothelium was stained 6 days later by intravenous injection of 200 μL fluorescent Bandeiraea simplicifolia lectin I, isolectin B4 at 0.1 mg/mL (Vector Laboratories, Burlingame, Calif.). Fifteen minutes later, the Matrigel plugs were excised and evaluated by confocal microscopy (Axiovert 100TV microscope; Carl Zeiss, Oberkochem, Germany; 40×/1.3 NA objective; and SPOT camera and software), and then lectin-fluorescein isothiocyanate (FITC) was extracted with RIPA lysis buffer (0.15 mM NaCl/0.05 mM Tris [tris(hydroxymethyl)aminomethane]-HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% sodium dodecyl sulfate) from 100-μg Matrigel plugs, to be quantified by fluorometry at 490 nm.
Statistical analysis. The statistical significance of differential findings between experimental groups and controls was determined by the Student t test. Findings were regarded as significant when 2-tailed P was less than 0.05.
Results
Minigenes encoded by expression vectors are expressed in mammalian cells. As demonstrated above, a DNA vaccine encoding the entire murine Flk-1 gene effectively induced CD8+ T cell-mediated antiangiogenesis that protected mice from tumor-cell challenge. Here, a minigene approach was utilized to demonstrate that Flk-1 epitopes can induce similar antiangiogenic responses as the whole Flk-1 gene vaccine in a C57BL/6J mouse model. To this end, 3 peptides were included in H-2Db- or H-2Kb-restricted minigenes based on the binding predicted for these MHC class 1 molecules using the HLA Peptide Binding Predictions program provided at the www website of the BioInformatics & Molecular Analysis Section (BIMAS) of the National Institutes of Health (NIH).
Expression vectors were constructed based on the backbone of pCMV/ER/Myc as described above. A HIVtat peptide (RKKRRQRRR, SEQ ID NO: 17), one of the commonly used membrane-translocating peptides, was also included in the minigene vaccine to facilitate the delivery of the encoded peptides. After transfection of 293 T cells with either pHI-myc, pHI-Dd-myc, or pHI-Kb-myc, correct expression of these constructs was demonstrated by Western blotting, which revealed single bands with the expected molecular mass of 15 kDa. The mature peptides did not contain the myc epitope because the vaccine vectors pHI, pHI-Db and pHI-Kb were generated by introducing a stop codon immediately downstream from the peptide-coding sequences. The correct vector constructs were confirmed by DNA sequencing. The empty pCMV vector was also included for control purposes.
The pHI-Db minigene vaccine protects mice against tumors of different origin by inducing immune responses that suppress tumor angiogenesis. Initially, the minigene DNA vaccines were evaluated in a prophylactic lung cancer model, where mice were first vaccinated with a minigene vaccine and then challenged intravenously with D121 lung carcinoma cells. In this case, the pHI-Db minigene elicited the best tumor protection, with 62.5% of mice surviving 75 days after tumor cell challenge. In contrast, none of the mice in the pCMV control group survived. The pHI or pHI-Kb vaccines induced only some tumor protection, however, with 25% of the mice surviving 75 days after tumor challenge.
To verify that the minigene vaccine effectively protects mice from tumors of different origins, the vaccine efficacy was also tested in a RM9 prostate carcinoma model. In this case, the pHI-Db minigene also protected the mice from RM9 tumor cell challenge indicating that the pHI-Db vaccine induces suppression of metastases independent of the tumor type.
In vivo depletion assays were performed to identify the cell population responsible for the tumor protection effects. Depletion of CD8+ cells completely abrogated the vaccine-induced protection, whereas the depletion of CD4+ cells moderately enhanced the protection against tumor challenge, indicating that the CD8+ T cells are the major effectors.
The specificity of the CTL responses was further investigated in 51Cr-release assays. The pHI-Db vaccine induced a specific cytotoxic response against a Flk-1+ endothelial cell line MS1, but not against Flk-1−RM9 prostate carcinoma cells. These results indicate that the cytotoxic response induced by the pHI-Db vaccine was indeed directed against endothelial cells, presumably specific for Flk-1, rather than against tumor cells. This finding also provides evidence that the pHI-Db minigene vaccine would induce an antiangiogenic response in vivo.
Matrigel assays were also performed, which indicated that vaccination with minigene pHI-Db indeed suppressed vascularization. This was clearly demonstrated by reduced blood vessel formation observed in representative Matrigel plugs after in vivo staining of the endothelium with FITC-conjugated lectin. This difference in vessel formation was also demonstrated quantitatively by measuring the average relative fluorescence. Taken together, these findings demonstrate that the pHI-Db minigene vaccine induced antiangiogenic effects, which protected mice from challenge with tumor cells of different origin.
The pHI-Db Vaccine Induces a Flk400-Specific Immune Response. To evaluate each of the 3 immunogenic Flk-1 peptide fragments encoded by the pHI-Db minigene, splenocytes isolated from mice immunized with pHI-Db were analyzed by ELISPOT assays using individual synthetic peptides as stimulators. A specific Flk400 response was detected only in the pHI-Db-vaccinated group of mice, whereas no significant Flk94- or Flk1210-specific responses were found in any of the experimental groups of mice, suggesting that Flk400 is the major epitope recognized by CTL effector cells.
To further confirm this observation, splenocytes from vaccinated mice were stimulated with synthetic peptides for 5 days and tested against MS1 and RM9 target cells in cytotoxicity assays. Only Flk400-stimulated splenocytes exhibited specific cytotoxic killing against Flk-1+MS1 target cells, but revealed almost no killing of Flk-1−RM9 tumor cells. Splenocytes stimulated with Flk94 induced low levels of MS1-specific killing whereas Flk1210-stimulated splenocytes mainly displayed low levels of nonspecific killing, confirming the dominance of the Flk400 epitope within the minigene vaccine.
When peptide-stimulated splenocytes isolated from pHI-Db-vaccinated mice were restimulated twice more in vitro with irradiated, peptide-loaded splenocytes every 7 days, and then tested again for their cytotoxicity, only those cells restimulated with Flk400-loaded splenocytes showed greatly enhanced cytotoxicity, resulting in a higher percent specific killing at a much lower effector-target (E/T) ratio. In contrast, such restimulation with Flk94-loaded splenocytes resulted in a lower level of MS1-specific killing, whereas cells restimulated with Flk1210-loaded splenocytes failed to induce any significant killing. Taken together, these findings indicate that restimulation with Flk400-loaded splenocytes enriches the CTL population that specifically targets Flk-1+ endothelial cells.
The tumor protective ability of a minigene vaccine encoding only Flk400 in the absence of Flk94 and Flk1210 was assessed and compared with the effect of the pHI-Db minigene vaccine in a EO771 breast carcinoma model. EO771 cells do not express Flk-1, but express surface H-2Db as detected by flow cytometry. In fact, both pHI-Db and pH1-Flk400 minigene vaccines significantly protected the mice against EO771 tumor cell challenge to an extent comparable to the protection induced by the DNA vaccine of Example 1 encoding the entire Flk-1 gene. The pHI-Db vaccine also achieved similar efficacy as Flk-1 whole-gene vaccine in RM9 prostate and D121 lung carcinoma models.
Long-term protection was established by the pHI-Db minigene vaccine, as well. At 10 months after their last vaccination, pHI-Db-vaccinated mice showed significantly reduced lung metastases after intravenous challenging with EO771 breast carcinoma cells, an Flk400-specific T cells could still be detected in the spleen of these mice.
The DNA Vaccine Encoding the Entire Elk-1 Gene Induces a Flk400-specific CTL Response. The Flk400-specific response was also induced by a DNA vaccine encoding the entire Flk-1 gene. Such responses were detected in splenocytes freshly isolated from pFlk-1-vaccinated mice as demonstrated by ELISPOT assays. These splenocytes maintained the specificity of the responses after in vitro stimulation with Flk400 peptides. However, controls were negative, because stimulation with Flk94 had no effect when compared to nonstimulated cells. Only nonspecific activation resulted from stimulation with Flk1210. Moreover, splenocytes isolated from pFlk-1-vaccinated mice also displayed preferential cytotoxic killing of EO771 tumor cells loaded with Flk400 as compared to the killing of unloaded EO771 cells. Similar results were also observed in pHI-Db-vaccinated mice, which were used as a positive control. The killing of EO771 or Flk400-loaded EO771 tumor cells was largely indistinguishable in pCMV or pHI control groups. Taken together, these findings strongly indicate that the DNA vaccine encoding the entire Flk-1 gene was capable of inducing a Flk400-specific immune response.
Numerous variations and modifications of the embodiments described above can be effected without departing from the spirit and scope of the novel features of the invention. It is to be understood that no limitations with respect to the specific embodiments illustrated herein are intended or should be inferred. It is, of course, intended to cover by the appended claims all such modifications as fall within the scope of the claims.

## Claims

1. A method of inhibiting endothelial cell proliferation in a mammal comprising the step of administering to the mammal an effective immunological response eliciting amount of a DNA composition comprising a DNA construct operably encoding a VEGF receptor polypeptide and a pharmaceutically acceptable carrier therefor, whereby said mammal exhibits an immune response elicited by vaccine and specific to proliferating endothelial cells, and wherein the DNA construct is operably incorporated in an attenuated bacterial vector.

2. The method of claim 1 wherein the mammal is a human.

3. The method of claim 1 wherein the VEGF receptor polypeptide is a full length protein selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

4. The method of claim 1 wherein the attenuated bacterial vector is selected from attenuated Salmonella typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus, BCG, Escherichia coli, Vibrio cholerae, and Campylobacter.

5. The method of claim 4 wherein the attenuated bacterial vector is an attenuated Salmonella typhimurium.

6. The method of claim 1 wherein the composition is administered orally.

7. The method of claim 1 wherein the VEGF receptor polypeptide comprises at least one immunogenic VEGF receptor fragment consisting of about 8 to 10 consecutive amino acid residues of a VEGF receptor selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

8. A method of inhibiting angiogenesis in a mammal comprising administering to said mammal an immunologically effective amount of a DNA composition comprising a DNA construct operably encoding a VEGF receptor polypeptide and a pharmaceutically acceptable carrier therefor, whereby said mammal exhibits an immune response elicited by vaccine and specific to proliferating endothelial cells, resulting in an inhibition of blood vessel formation, and wherein the DNA construct is operably incorporated in an attenuated bacterial vector.

9. The method of claim 8 wherein the mammal is a human.

10. The method of claim 8 wherein the VEGF receptor polypeptide is a full length VEGF receptor selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

11. The method of claim 8 wherein the VEGF receptor polypeptide comprises at least one immunogenic VEGF receptor fragment consisting of about 8 to 10 consecutive amino acid residues of a VEGF receptor selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

12. The method of claim 8 wherein the attenuated bacterial vector is selected from attenuated Salmonella typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus, BCG, Escherichia coli, Vibrio cholerae, and Campylobacter.

13. The method of claim 8 wherein the attenuated bacterial vector is an attenuated Salmonella typhimurium.

14. The method of claim 8 wherein the vaccine is administered orally.

15. A method of inhibiting tumor growth in a mammal comprising administering to said mammal an immunologically effective amount of a DNA composition comprising a DNA construct operably encoding a VEGF receptor polypeptide and a pharmaceutically acceptable carrier therefor, whereby said mammal exhibits an immune response elicited by vaccine and specific to proliferating endothelial cells, resulting in the arresting of tumor growth, reduction in tumor size, or inhibition of tumor dissemination and wherein the DNA construct is operably incorporated in an attenuated bacterial vector.

16. The method of claim 15 wherein the mammal is a human.

17. The method of claim 15 wherein the VEGF receptor polypeptide is a full length VEGF receptor selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

18. The method of claim 15 wherein the VEGF receptor polypeptide comprises at least one immunogenic VEGF receptor fragment consisting of about 8 to 10 consecutive amino acid residues of a VEGF receptor selected from the group consisting of VEGFR-2 (KDR; SEQ ID NO: 2), VEGFR-1 (Flt-1; SEQ ID NO: 4), and Flk-1 (SEQ ID NO: 6).

19. The method of claim 15 wherein the attenuated bacterial vector is selected from attenuated Salmonella typhimurium, Salmonella typhi, Shigella, Bacillus, Lactobacillus, BCG, Escherichia coli, Vibrio cholerae, and Campylobacter.

20. The method of claim 15 wherein the attenuated bacterial vector is an attenuated Salmonella typhimurium.

21. The method of claim 15 wherein the vaccine is administered orally.

22. The method of claim 15 wherein the attenuated bacterial vector is an attenuated Salmonella typhi.

23. The method of claim 1 wherein the attenuated bacterial vector is an attenuated Salmonella typhi.

24. The method of claim 8 wherein the attenuated bacterial vector is an attenuated Salmonella typhi.