KR20010102556A - Compositions and methods for treating cancer and hyperproliferative disorders - Google Patents

Abstract

Provided are compositions and methods that are effective for eliciting immune responses to inhibit endothelial cell proliferation and angiogenesis associated with abnormal or unwanted cell proliferation, particularly angiogenesis and tumor growth. The composition comprises natural or synthetic proteins, peptides or protein fragments containing all or an active part of the growth factor in a pharmaceutically acceptable carrier. Preferred growth factors include basic fibroblast growth factor and vascular endothelial growth factor. The method comprises administering a composition described herein to a human or animal in a dosage sufficient to elicit an immune response. The method is particularly useful for treating diseases and processes that are mediated by undesired and uncontrollable cell proliferation such as cancer when unregulated cell proliferation is affected by the presence of growth factors. Administration of the composition to humans or animals with metastasized tumors is useful for preventing the growth or expansion of such tumors.

Description

Therapeutic composition and method of treatment for cancer and hyperproliferative disorders {COMPOSITIONS AND METHODS FOR TREATING CANCER AND HYPERPROLIFERATIVE DISORDERS}

Cross Reference of Related Application

The present invention is a non-preliminary application of U. S. Provisional Patent Application No. 60 / 077,460, filed Mar. 10, 1998, filed U.S. Part of the continuing application of patent application 09 / 264,213, filed U.S. Part of the ongoing application of patent application 09 / 266,453, which claims priority.

Although many cancers are treatable with chemotherapeutic agents, many cancers are inherently tolerated and the rest will acquire resistance during or after chemotherapy. Cancer is often resistant to more than one type of drug. This phenomenon is called compound tolerability or MDR. As a result, there is a great need for compositions and methods that can be used in combination with, or as an alternative to, chemotherapy for the treatment of cancer.

The main clinical problem of cancer is metastasis. Until the primary tumor is identified and localized, the seed cells often escape and migrate or metastasize to other organs in the body where they settle into secondary tumors. Surgical procedures are almost insufficient to cure cancer because complex secondary tumors survive and multiply after the primary tumor is removed. As a result, there is an immediate and persistent demand for the eradication of existing secondary tumors.

Cancer cells that have exited the primary tumor typically migrate into the venous and lymphatic circulation until they stay on the downstream capillary or lymph nodes. However, only one out of 10,000 cancer cells that escaped the primary tumor survive and settle into secondary tumors. Successful cancer cells have found the right environment for survival and growth. Suitable environments include hormones and growth-promoting factors. Stimulators include local growth factors, hormones produced by the host, and self-stimulated growth factors produced by the tumor cells themselves. As a result, there is a continuing and demanding need for techniques that can prevent or inhibit cancer metastasis and the formation of secondary tumors.

In addition, many other hyperproliferative disorders exist. Hyperproliferative disorders are caused by non-cancerous (ie non-tumoral) cells that are overproduced in response to certain growth factors. Examples of such hyperproliferative disorders include diabetic retinopathy, psoriasis, endometriosis, macular degeneration and benign growth disorders such as prostatic hypertrophy and lipoma.

It is known that many new cancers are caused by growth factors (i.e., angiogenesis factors) that affect the cancer cells themselves or normal tissues around the cancer that promote cancer cell survival and stimulate existing cancers and hyperproliferative disorders. . There is a direct correlation between the growth of certain growth factors and cancer proliferation. Potential treatments will be to control the levels of circulating growth factors in the patient to prevent the onset or recurrence of cancer and to weaken or eliminate existing cancer. Therefore, there is a need for compositions that remove target growth factors from the circulation or inhibit growth-promoting activity of growth factors.

Cell proliferation is a common process in all living organisms and involves a number of factors and signals that elaborately balance to maintain a regular cell cycle. The general process of cell division consists of a series of two processes: nuclear fission (mitosis) and cytoplasmic division (cytoplasm). Because organisms are cells that continue to grow and replace, cell proliferation is a key process essential for the normal functioning of almost all biological processes. Whether or not a mammalian cell will grow and divide is determined by a variety of feedback control mechanisms, including the availability of space in which the cell can grow and the secretion of specific stimuli and inhibitors in the immediate environment.

If normal cell proliferation is interrupted or somewhat disrupted, the result can affect the array of biological functions. Disruption of proliferation can be due to a number of factors, such as various signaling chemicals, the absence of growth factors or overheating or modification of the environment. Some disorders characterized by abnormal cell proliferation include cancer, abnormal development of embryos, inadequate formation of antibodies, difficulty in wound fusion and dysfunction of inflammatory and immune responses.

Cancer is characterized by abnormal cell proliferation. Cancer cells often exhibit several properties that are harmful to the host, including the ability to invade other tissues and induce capillary invasion to ensure sufficient blood supply to proliferating cancer cells. One of the defining characteristics of cancer cells is that they abnormally respond to regulatory mechanisms that regulate the division of normal cells and continue to divide in a relatively unregulated manner until they kill the host.

Angiogenesis and angiogenesis related diseases are closely affected by cell proliferation, and thus cytokines and growth factors. As used herein, the term “angiogenesis” refers to neovascularization into tissues or organs. Under normal physiological conditions, humans or animals progress angiogenesis only in very specific limited circumstances. For example, angiogenesis is commonly observed in wound fusion, development of fetuses and embryos, and in the formation of antibodies, endometrium and placenta. The term "endothelium" is defined herein as a thin film of squamous cells lining serous, lymphatic and blood vessels. These cells are defined herein as "endothelial cells". The term "endothelial inhibitory activity" generally means the ability of a molecule to inhibit angiogenesis. Inhibition of endothelial cell proliferation also results in inhibition of angiogenesis.

Both controlled and unregulated angiogenesis are thought to proceed in a similar manner. Endothelial and envelope cells surrounded by the basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes secreted by endothelial cells and leukocytes. Endothelial cells lining the lumen of blood vessels then expand through the basement membrane. Angiogenic stimulants induce endothelial cells to migrate through the eroded basement membrane. The migrating cells form "sprouts" from the mother blood vessels where endothelial cells mitosis and proliferate. Endothelial sprouts form a capillary loop that merges with one another to produce renal vessels.

Persistent, unregulated angiogenesis is a cause of pathological damage occurring in and manifesting in the symptoms of various diseases, tumor metastasis and abnormal growth by endothelial cells. Symptoms of various pathological diseases in which unregulated angiogenesis occurs are classified as angiogenesis-dependent, angiogenesis-associated or angiogenesis-related diseases. These diseases are the result of abnormal or unwanted cell proliferation, in particular endothelial cell proliferation.

The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971 by Judah Folkman (N. Engl. Jour. Med. 285: 1182 1186, 1971). In its simplest sense, this hypothesis suggested that expansion of tumor volume above a certain phase requires the induction of neocapillaries. For example, micrometastasis of the lung in the early prevascular phase of mice will not be detectable except by high performance microscopy on histological sections. In addition, indirect evidence explaining the concept that tumor growth is angiogenesis dependent is described in U.S. Patents 5,639,725, 5,629,327, 5,792,845, 5,733,876 and 5,854,205, all of which are incorporated herein by reference.

Recent research in the field of angiogenesis has led to a recent paradigm explaining that phenotypic changes involving tumor-associated angiogenesis are mediated by angiogenic switches. In the homeostatic state, the balance between the angiogenesis inhibitor and the stimulant is maintained. Conversely, during tumor formation, this balance shifts to the angiogenic phenotype. This switch may be mediated by upregulation of endogenous angiogenic stimulants such as fibroblast growth factor, vascular endothelial growth factor or interleukin or downregulation of angiogenesis inhibitors such as angiostatin or endostatin proteins. .

One example of a disease mediated by angiogenesis is neovascular disease of the eye. The disease is characterized by invasion of the constituent tissues of the eye, such as the retina or cornea of the neovascular system. This is the most common cause of blindness and is involved in approximately 20 eye diseases. In age-related macular degeneration, a related visual problem is caused by intraocular proliferation of choroidal capillaries through defects in the Burk's membrane with the proliferation of fibrovascular tissue under epithelial fragments of the retina. Angiogenesis damage is also associated with diabetic retinopathy, prematurity retinopathy, corneal graft rejection, neovascular glaucoma and posterior capsular fibrosis. Other diseases associated with angiogenesis of the cornea include epidemic keratitis, vitamin A deficiency, contact lens overdose, atopic keratitis, epicondylar keratitis, pterygium dry keratitis, Sjogren's syndrome, rosacea, phylectenulosis, syphilis, mycoa Bacterial infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, herpes simplex infections, shingles infections, protozoan infections, Kaposi's sarcoma, Muren's ulcers, terryen degenerate degeneration, marginal exfoliation, rheumatoid arthritis, systemic lupus , Polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens Johnson disease, and periphigoid radial corneal incision.

Diseases related to retinal / choroidal neovascularization include diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoidosis, syphilis, fibrosulfoma, papette disease, vein occlusion, arterial occlusion, carotid atresia, chronic uveitis / vitreous disease , Mycobacterial infection, Lyme disease, Systemic lupus erythematosus, Prematurity retinopathy, Ills disease, Behcet's disease, Retinitis or choroiditis, Infection ocular histoplasmosis, Best disease, Myopia, Siwa, Stagart disease, Peripheral uveitis, chronic retinal detachment, high viscosity syndrome, toxoplasmosis, trauma and postoperative laser complications, but are not limited thereto. Other diseases include, but are not limited to, diseases related to cutaneous redness (anterior angiogenesis) and disorders due to abnormal proliferation of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy.

Another disease believed to be involved in angiogenesis is rheumatoid arthritis. Vasculature of the lining of the joint secreting synovial fluid proceeds angiogenesis. In addition to the formation of a renal network, endothelial cells release reactive oxygen species and factors that induce pannus growth and cartilage damage. Factors involved in angiogenesis may actively contribute to and maintain a chronic inflammatory state of rheumatoid arthritis.

Factors associated with angiogenesis may also play a role in osteoarthritis. Activation of chondrocytes by angiogenesis-related factors contributes to joint damage. Later, angiogenic factors will promote renal bone formation. Treatment interventions that prevent bone damage can stop disease progression and provide relief for patients with arthritis.

Chronic inflammation may also be associated with pathological angiogenesis. Symptoms of diseases such as ulcerative colitis and Crohn's disease show histological changes due to internal growth of neovascularization into inflammatory tissue. Bartonellaosis, a bacterial infection found in South America, can lead to chronic symptoms characterized by the proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. Plaques formed in the lumen of blood vessels are shown to have angiogenic stimulatory activity.

One of the most common angiogenic diseases in children is hemangioma. In most cases, the tumor is benign and degenerates without intervention. In more severe cases, the tumor proceeds in large spongy and invasive forms and leads to clinical complications. Hemangioma, a systemic form of hemangioma, has a high mortality rate. There are therapeutic resistant hemangiomas that cannot be treated with the currently used therapies.

Angiogenesis is also responsible for the damage found in hereditary diseases such as Osler-Weber-Rang du disease or hereditary hemorrhagic capillary dilator. It is a hereditary disease characterized by tumors of complex small hemangiomas, blood vessels or lymphatic vessels. Hemangiomas are often found in the skin and mucosa, accompanied by nasal hemorrhage (nose) or gastrointestinal bleeding, sometimes pulmonary or hepatic arteriovenous bleeding.

It is therefore clear that cell proliferation, in particular endothelial cell proliferation, plays an important role in the pathology and progression of cancer growth and metastasis. If this abnormal or unwanted proliferative activity can be suppressed, suppressed or eliminated, it will not grow even if the tumor is present. In the symptoms of the disease, abnormal or unwanted cell proliferation and inhibition of angiogenesis can prevent the damage caused by invasion of the neoplastic microvascular system. Treatment aimed at regulating the cell proliferation process can lead to the elimination or alleviation of such diseases.

Accordingly, what is needed are compositions and methods that can inhibit abnormal or unwanted cell proliferation associated with tumors. The composition should be able to suppress the onset of the disease and the growth of the tumor by overwhelming the activity of the endogenous growth factor of the pretransitional tumor and preventing the spread of cancerous cells. The composition should also be able to control the formation of capillaries during angiogenesis. Finally, the compositions and methods for inhibiting cell proliferation should preferably be non-toxic and cause little side effects.

Summary of the Invention

The present invention generally includes methods and compositions for preventing or treating cancer. More specifically, the present invention encompasses immunogen growth factor-containing compositions comprising growth factors or active fragments thereof, optionally in combination with a delivery vehicle. Without being bound by the theory below, the compositions of the present invention can elicit a cellular or immune response that results in the prevention and elimination of cancer.

The present invention provides methods for vaccination of humans or animals against growth factors associated with certain types of cancer and hyperproliferative disorders. Certain cancers are associated with only one growth factor, while others are controlled by multiple growth factors. For example, certain T cell lymphomas produce growth factor IL-2, which promotes proliferation through autosecretory action; Other tumors produce factors that stimulate angiogenesis and stimulate the growth of metastatic cancer lesions by inducing tissue angiogenesis at the site of metastasis.

Growth factor-containing compositions of the present invention are delivery or delivery vehicles such as liposomes or vesicles having growth factors, growth factor fragments, synthetic peptides of specific epitopes of growth factors, or modified growth factor fragments added to their outer surfaces. It may optionally include. In alternative embodiments, the growth factor or immunogen fragments thereof may be partially or wholly encapsulated in a carrier such as liposomes. In another alternative embodiment of the invention, the growth factor or active portion thereof may be transported to the desired site by a delivery mechanism involving the use of colloidal metals such as colloidal gold. The aforementioned compositions are useful as vaccines that otherwise induce immunity to growth factors that are “self” recognized by the immune system and are not naturally antigenic. The composition is further useful in therapy to mitigate the proliferation of tumor cells. Without being bound by the following theory, it is believed that the resulting circulating antibodies bind to growth factors to prevent the onset of cancer proliferation, to eliminate existing cancer or to inhibit the spread of cancer.

The invention also encompasses isolated recombinant antibodies specific for growth factors. Isolated antibodies are produced by and purified from humans or animals with a strong immune system and injected into humans or animals with weak or non-functional immune systems requiring such circulating antibodies. Thus, according to the present invention cancer is eliminated or inhibited by active immunization of patients using antigenic growth factor-containing compositions or by passive immunization via administration of antibodies or groups of antibodies specific for growth factor epitopes. In addition, the patient is immunized with a growth factor composition prior to relapse after the onset or treatment of cancer.

It is therefore an object of the present invention to provide methods and compositions for removing cancer and inhibiting tumor growth in humans or animals suffering from cancer.

It is another object of the present invention to provide methods and compositions for treating and preventing the onset or spread of cancer.

It is a further object of the present invention to provide methods and compositions for removing cancer and inhibiting tumor growth of humans or animals with cancer by eliciting active cellular and / or humoral responses in the host.

It is another object of the present invention to provide methods and compositions for eliminating and preventing the development of hyperproliferative disorders.

It is another object of the present invention to provide methods and compositions for vaccination of humans or animals for selected growth factors.

It is another object of the present invention to provide methods and compositions for passive immunization of humans or animals against selected growth factors.

It is another object of the present invention to provide a growth factor-containing composition which is antigenic and which elicits an immune response against a sex factor in humans or animals.

Another object of the present invention is to provide a vaccine composition comprising a growth factor and a carrier which are non-immunogens in a human or animal immunized with the composition, wherein the growth factor is directed to the growth factor when the composition is administered to a human or animal. It is added to the surface of the carrier to be an immunogen against.

Another object of the present invention is the growth factor fibroblast (FGF), interleukin, keratin synthesis cell growth factor, colony stimulating factor, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), conversion growth factor, Schwann cells Induced growth factor, nerve growth factor (NGF), platelet-induced growth factor (PDGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), glial growth factor, tumor necrosis factor, prolactin, growth It is to provide a growth factor-containing composition comprising a hormone and / or active fragments thereof.

It is yet another object of the present invention to provide growth factor peptide fragments modified with antigenic residues to enhance the patient's response to growth factors and methods of use thereof.

It is another object of the present invention to provide a growth factor-containing composition wherein the carrier for growth factor comprises liposomes.

Another object of the present invention is to provide a growth factor-containing composition wherein the carrier for the growth factor comprises a colloidal metal.

Another object of the present invention is to provide a growth factor containing composition wherein the carrier is a baculovirus-derived vesicle.

Another object of the present invention is to provide a growth factor peptide fragment-containing composition in combination with a pharmaceutically acceptable adjuvant for stimulating an immune response.

Another object of the present invention is hemangioma, solid tumor, blood-mediated tumor, leukemia, metastasis, capillary dilator, psoriasis, scleroderma, purulent granulomas, myocardial angiogenesis, Crohn's disease, plaque angiogenesis, arteriovenous malformation, corneal disease , Skin redness, neovascular glaucoma, diabetic retinopathy, posterior capsular fibrosis, arthritis, diabetic angiogenesis, macular degeneration, wound union, peptic ulcer, Helicobacter related disease, fracture, keloid, angiogenesis, hematopoiesis, ovulation, menstruation, Provided are therapeutic compositions and methods for diseases and processes mediated by angiogenesis, including but not limited to placental formation and cerebellar fever.

Another object of the present invention is to provide anti-growth factor antibodies useful for the passive immunization of humans or animals against growth factors.

Another object of the present invention is to provide a growth factor-containing composition which can be administered intramuscularly, intravenously, transdermally, orally or subcutaneously.

These and other objects, features and advantages of the present invention will become apparent after a detailed description of the embodiments set forth below and the reading of the appended claims.

The present invention relates to methods and compositions for preventing or eliminating cancer in humans or animals. In more detail, the present invention relates to immunogenic compositions comprising growth factors, active fragments thereof, antibodies specific for growth factors, and methods of use thereof.

1A is a graph showing the results of experiments performed to illustrate growth inhibition of experimental lung metastases. Mice pretreated with L (HBD) (bFGF peptide from heparin-binding domain conjugated to liposomes), L (RBD) (receptor binding domain of bFGF conjugated to liposomes), L (LA) (empty liposomes) or PBS Is intravenously challenged with 5 × 10 ⁵ B16BL6 tumor cells. Mice are beheaded 14 days after inoculation, lungs are removed and lung metastases counted using an anatomical microscope. Tumor growth was evident in all groups analyzed. Mice treated with L (HBD) showed a 96% inhibition of lung metastasis.

1B is a graph showing the results of experiments performed to illustrate the inhibition of transitions for LLC-LM experiments. Mice pretreated with L (HBD) or L (LA) are challenged intravenously with 5 × 10 ⁵ LLC-LM tumor cells. Mice are beheaded 17 days after inoculation, lungs are removed and lung weights are measured. Mice treated with L (HBD) inhibited the development of lung metastasis by 9S%.

FIG. 2 is a photograph comparing the differences in dorsal B16BL16 metastasis from mice treated with liposome lipid A (top) (control) and mice treated with bFGF peptide from the heparin-binding domain (bottom).

3 is a photograph comparing the efficacy of vaccination with peptides from the heparin-binding domain of bFGF in mouse lungs to the efficacy of control vaccination in lung B16BL6 growth and development.

FIG. 4 shows serum from mice vaccinated with heparin binding domain peptide of bFGF with total bFGF (white square), heparin binding domain peptide bFGF (white diamond), compared to normal mouse serum (NMS) (white circle). This graph shows the reactivity.

5 is a graph showing the reactivity of serum from mice vaccinated with the receptor binding domain peptide of bFGF with total bFGF (white square), receptor binding domain peptide (white diamond) and NMS (white circle).

6A shows histological analysis of gelatin sponges removed from mice vaccinated with PBS control, LLA control, heparin binding domain peptide of bFGF and receptor binding domain peptide of bFGF. As further described in Example 9, gelatin sponges containing recombinant human bFGF were replaced with liposome lipid A control, heparin binding domain peptides to determine if vaccination against bFGF molecules affected bFGF-induced angiogenesis. Vaccinated with liposomes containing, liposomes containing receptor binding domain peptides, or PBS, and then transplanted into the left mesenchyme of mice. 6B-6D show the histological analysis of the experiments performed to demonstrate inhibition of FGF-2-induced angiogenesis. Gelatin sponges containing FGF-2 are transplanted into the liver of mice pretreated with L (HBD), L (RBD) or L (LA). After 14 days of implantation, the sponges are removed and cut for tissue analysis. (FIGS. 6B and 6C) Hematoxylin and eosin (H & E) staining of sponges from mice treated with L (LA) or L (RBD) showed cell infiltration and angiogenesis. (d) H & E staining of sponges from mice treated with L (HBD) showed a significant decrease in cell infiltration and angiogenesis.

7 shows histological analysis of gelatin sponges removed from mice vaccinated with PBS controls, LLA controls, liposomes containing heparin binding domain peptides of bFGF and liposomes containing receptor binding domain peptides of bFGF. As further described in Example 9, to determine if vaccination can inhibit tumor-induced angiogenesis, gelatin sponges containing B16BL6 melanoma cells were treated with control liposomes (liposomal lipid A), heparin binding Mice are transplanted to the liver after vaccination with liposomes bound to the domain peptide, liposomes bound to the receptor binding domain peptide, or PBS.

8 shows bFGF stimulated proliferation of HUVECs in the presence of bFGF peptides: control peptide (black circle), heparin binding domain peptide of bFGF (white circle) and receptor binding domain peptide of bFGF (white square).

Figure 9 shows the reproducibility of the effect shown in Figure 8: bFGF stimulated proliferation of HUVEC in the presence of bFGF peptide, heparin binding domain peptide of bFGF (white circle) and receptor binding domain peptide of bFGF (white square).

FIG. 10 is a bar graph showing the relative effects of bFGF heparin binding domain peptide (HEP) or bFGF receptor binding domain peptide (REC) on the growth of B16BL6 tumor cells in vitro.

FIG. 11 is a bar graph showing the inhibition of growth and development of LLC-LM metastasis in mice vaccinated with liposomes containing the heparin binding domain peptide of bFGF.

12 (a) and 12 (b) provide graphs showing anti-bFGF titers in serum from mice vaccinated with bFGF-liposomes or control liposomes. Data provided by the graph was collected at day 35, pooled, serially diluted and then analyzed for serum reactivity from the group of five vaccinated mice to analyze reactivity to total bFGF by measuring absorbance in an ELISA assay. Indicates.

FIG. 13 is a graph showing anti-bFGF titers in serum from each mouse vaccinated with bFGF-liposomes. As described in Example 11, C57BI / 6J mice are vaccinated with liposomes containing bFGF according to the protocol outlined in the method section. Serum from each mouse labeled B1-B5 is collected on day 35, serially diluted and analyzed for reactivity to total bFGF by measuring absorbance in ELISA assays. Serum reactivity from mice vaccinated with liposome controls is not different from NMS. The data shown correspond to the method and protocol of Example 11 as follows: NMS (dotted square), mouse 1 (black diamond), mouse 2 (black square with white dot), mouse 3 (white diamond); Mouse 4 (black square) and Mouse 5 (white square).

14 is a graph showing the size of primary tumors after 14 days of challenge in mice vaccinated with bFGF or control liposomes. As described in Example 11, Balb / cByJ mice are vaccinated with liposomes containing bFGF (bFGF) or liposomes without bFGF (control) according to the protocol outlined in the method paragraph. On day 35 mice are subcutaneously challenged with 1 × 10 ⁶ LLC-LM on the back. Data shown represent tumor volume measured 14 days after challenge of each mouse in each vaccination group.

FIG. 15 is a bar graph showing the size of primary tumors after 14 days of challenge in mice vaccinated with bFGF or control liposomes. As described in Example 11, groups of five Balb / cByJ mice were vaccinated with liposomes containing bFGF (bFGF liposomes), liposomes without bFGF (control liposomes) or with PBS, according to the protocol outlined in the method section. Process (PBS). On day 35 mice are subcutaneously challenged with 1 × 10 ⁶ LLC-LM on the back. Data shown is 1 SD of mean tumor volume ± mean, measured 14 days after challenge in each of the 3 vaccination groups.

16 (a)-(c) are graphs showing the immunoreactivity of test peptides against autologous peptide fragments of VEGF. As described in Example 13, Balb / cByJ mice are vaccinated with liposomes containing VEGF peptide 1-3 introduced into liposomes by conjugation according to the protocol outlined herein. Serum from each mouse is collected on day 35, and serially diluted and analyzed for reactivity of VEGF with autologous peptide fragments by measuring absorbance in ELISA assays. NMS controls show the reactivity of serum from non-vaccinated mice and do not differ from the reactivity of serum from mice vaccinated with liposome controls.

17 (a) and 17 (b) are graphs showing the immunoreactivity of test peptides against autologous peptide fragments of VEGF. As described in Example 13, Balb / cBYJ mice are vaccinated with liposomes containing VEGF peptides 2 and 3 introduced into liposomes by simple encapsulation according to the protocol outlined herein. Serum from each mouse is collected on day 35, and serially diluted and analyzed for reactivity of VEGF with autologous peptide fragments by measuring absorbance in ELISA assays. NMS controls show the reactivity of serum from non-vaccinated mice and do not differ from the reactivity of serum from mice vaccinated with liposome controls.

FIG. 18 is a graph comparing the inhibitory effect on the growth and development of B16BL6 experimental metastases in mice vaccinated with lipid liposome A, VEGF peptide SEQ ID NO: 6 and bFGF heparin binding domain peptide.

FIG. 19 provides a graph comparing the inhibitory potency of peptides corresponding to heparin binding domain or receptor binding domain of FGF-2 when applied to HUVEC in the presence of FGF-2, 5 ng / ml in 72 hour proliferation assay. Both heparin binding (O) and receptor binding (□) domain peptides inhibit HUVEC proliferation in a dose dependent manner. Each point represents the mean +/- 1 standard deviation.

20 is a graph showing the results of experiments performed to demonstrate induction of antibody responses to FGF-2 heparin binding domain peptides. Mice are treated with FGF-2 peptide covalently bound to liposome vesicles every two weeks for six weeks. Serum from mice treated with heparin binding domain peptides responds to FGF-2 heparin binding domain peptides (◇) and native FGF-2 molecules (□) in ELISA. This serum did not show immunoreactivity against receptor binding domain peptide (○) or other VEGF (Δ).

21 ac shows inhibition of B16BL6 tumor growth and angiogenesis in liver sponge inserts. Gelatin sponges containing 5 × 10 ⁴ B16BL6 tumor cells are transplanted into the liver of mice pretreated with L (HBD), L (RBD) or L (LA). After 14 days of implantation, the sponges are removed and cut for tissue analysis. H & E staining of sponges removed from mice treated with L (LA) (a) or L (RBD) (b) showed B16BL6 tumor growth and angiogenesis, while sponges removed from mice treated with L (HBD) (c) Contains abundant red blood cells lacking the defined structure surrounding them.

The invention may be more readily understood by reference to the following detailed description of the specific embodiments contained herein. Although the present invention has been described with reference to specific details of specific embodiments thereof, these details should not be construed as limiting the scope of the invention. The entire text of the references cited herein are hereby incorporated by reference in their entirety and described in U.S. Pat. Patent applications 09 / 266,453 and 09 / 264,213, US preliminary application 60 / 077,460 and US patent 5,919,459.

The present invention includes methods and compositions for preventing or eliminating cancer and hyperproliferative disorders of humans or animals. The immunogen growth factor-containing compositions of the present invention are combined with growth factors containing carriers such as liposomes and vesicles, immunogen growth factors and peptide fragments thereof, immunogen growth factor peptide fragments in combination with adjuvants, modified growth factor peptide fragments, adjuvants Modified growth factor peptide fragments, growth factor peptide fragments containing carriers and modified growth factor peptide fragments containing carriers. In addition, the present invention includes antibodies and other cellular responses specific for growth factors. The present invention also provides the use of ribonucleic acid sequences encoding such proteins and ribonucleic acid sequences for transfecting humans or animals to induce an immune response to antigenic growth factors in the body to introduce antigenic proteins into the system. It includes.

Justice

The terms "a", "an" and "the" as used herein are defined to mean one or more and include the plural unless inadequate in the text.

The term “antibody (s)” as used herein includes monoclonal antibodies, polyclonal, chimeric, single-chain, bispecific, monkey and humanized antibodies and Fab fragments, including the products of Fab immunoglobulin expression libraries. do.

The term “antigen” refers to all or fragments thereof capable of inducing an immune response in an animal. The term includes regions that are responsible for immunogenicity and antigenic or antigenic determinants.

As used herein, the term "soluble" means partially or completely dissolved in an aqueous solution.

As used herein, the phrase “biological activity” refers to the functionality, reactivity and specificity of a compound derived from a biological system or a compound reactive with them or other compounds that mimic the functionality, reactivity and specificity of these compounds. Examples of suitable biologically active compounds include enzymes, antibodies, antigens and proteins.

The term "body fluid", as used herein, refers to saliva, gingiva secretion, cerebrospinal fluid, gastrointestinal fluid, mucus, genitourinary secretion, lubricating fluid, blood, serum, plasma, urine, cystic fluid, lymph, ascites, pleural effusion, interstitial fluid, Intracellular fluids, eye fluids, semen, milky fluids, vitreous fluids and nasal fluids.

The phrase “consist essentially of” is used herein to exclude elements that will substantially modify the essential nature of the peptides mentioned by the phrase. Thus, the description of a peptide consisting essentially of excludes amino acid substitutions, additions or deletions that substantially modify the biological activity of the peptide.

The term "peptide" is a chain of amino acids (typically L-amino acids) to which alpha carbon is bound via a peptide bond formed by a condensation reaction between a carboxyl group of an alpha carbon of one amino acid and an amino group of an alpha carbon of another amino acid. . One terminal amino acid of the chain (ie, amino terminus) has a free amino group, while the other terminal amino acid of the chain (ie, carboxy terminus) has a free carboxyl group. Likewise, the term "amino terminus" (abbreviated to N-terminus) refers to the free alpha-amino group of amino acids at the amino terminus of the peptide or the alpha-amino group of amino acids at other positions in the peptide (imino group when involved in peptide binding). To mention. Likewise, the term “carboxy terminus” (abbreviated to C-terminus) refers to a free carboxyl group of amino acids at the carboxy terminus of a peptide or a carboxyl group of amino acids at another position in the peptide.

Typically, the amino acids that make up the peptide are numbered in order, starting from the amino terminus and increasing in number toward the carboxy terminus of the peptide. Thus, when one amino acid is "followed" another amino acid, the amino acid is located closer to the carboxy terminus of the peptide than the preceding amino acid.

The term "residue" refers to an amino acid (D or L) or amino acid replica introduced into an oligopeptide by an amide bond or an amide bond mimetic. Likewise, an amino acid can be a natural amino acid or can include known analogues (ie, amino acid replicas) of natural amino acids that act in a similar manner to natural amino acids, unless otherwise defined. Amide binding mimetics also include peptide backbone modifications well known to those skilled in the art.

In addition, the skilled person may, as described above, replace, delete or delete each amino acid or a small proportion of amino acids (typically 5% or less, more typically 1% or less) in the encoded sequence, and add or delete one or more amino acids. It will be appreciated that the addition is a conservative modified modification that results in the substitution of amino acids by chemically similar amino acids. Conservative substitution tables that provide functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) aspartic acid (D), glutamic acid (E);

3) asparagine (N), glutamine (Q);

4) arginine (R), lysine (K);

5) isoleucine (I), leucine (L), methionine (M), valine (V); And

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The phrase "isolated" or "biologically pure" refers to a substance that is substantially or essentially free of components that typically accompany it, as found in its native state. Thus, the peptides described herein do not contain substances that are commonly associated with the field environment. Typically, the isolated, antiproliferative peptides described herein are at least about 80%, typically at least about 90%, preferably at least about 95% pure, as measured by band intensity on gels stained with silver.

Protein purity or homogeneity can be manifested by a number of methods well known in the art, such as polyacrylamide gel electrophoresis of protein samples, followed by visualization by staining. High resolution will be required for certain purposes and HPLC or similar means is used for purification.

If the proteins and peptides of the invention are relatively short in length (ie up to about 50 amino acids), they are often synthesized using standard chemical peptide synthesis techniques.

Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by the continuous addition of the remaining amino acids of the sequence is a preferred method for chemical synthesis of the antiproliferative peptides described herein. Solid phase synthesis techniques are known to those skilled in the art.

In contrast, the antigenic peptides described herein are synthesized using recombinant nucleic acid methodology. Generally, this produces a nucleic acid sequence encoding a peptide, places the nucleic acid in an expression cassette under the control of a specific promoter, expresses the peptide in the host, isolates the expressed peptide or polypeptide, and then denatures the peptide if necessary. Steps. Sufficient technology is found in patented technology to teach this process to the skilled person.

Once expressed, the recombinant peptides can be purified according to standard procedures, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Substantially pure compositions having about 50-95% homogeneity are preferred, with 80-95% or more homogeneity being most preferred for use as a therapeutic agent.

Those skilled in the art will appreciate that, after chemical synthesis, biological expression, or purification, the immunogenic peptide may have a structure that is substantially different from the native structure of the constituent peptide. In this case, it is often necessary to denature and reduce the immunogen peptides and then allow the peptides to re-fold into the desired structure. Methods of reducing and denaturing proteins and inducing refolding are well known to those skilled in the art.

As used herein, the term “growth factor” refers to growth factors and polypeptide angiogenesis factors and modified derivatives and active peptide fragments thereof. The term "growth factor" is:

Fibroblast growth factor (FGF);

Interleukin 1-12 (IL-1α, β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12);

Keratin synthesis cell growth factor;

Colony stimulating factors such as granulocyte colony stimulating factor (G-CSF), macrophage colony forming stimulating factor (M-CSF or CSF-1) and GM-CSF;

Epidermal growth factor (EGF);

Vascular endothelial growth factor (VEGF, otherwise known as vascular permeability factor);

Conversion growth factor α (TGF-α);

Conversion growth factors β1 to β5 (TGF-β);

Schwann cell-induced growth factor;

Nerve growth factor (NGF);

Platelet-induced growth factor (PDGF);

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2);

Glial growth factor;

Tumor necrosis factors α and β (TNF-α, TNF-β);

Prolactin;

Prostaglandins; And

Contains growth hormones.

As used herein, the term “immunogen” refers to a substance that elicits or increases the production of antibodies, T-cells, and other reactive immune cells against endogenous growth factors and elicits an immune response in humans or animals.

The subject may have circulating antibodies against endogenous growth factors, but the subject did not undergo an immune response to the growth factors. Thus, the term “non-immunogenic”, as used herein, typically does not elicit circulating antibodies, T-cells or reactive immune cells at all or only at minimal levels and does not normally elicit an immune response against itself in an individual. Refers to endogenous growth factors in the native state. The immune response occurs when the individual produces sufficient antibodies, T-cells and other reactive immune cells to the injected growth factor compositions of the present invention to modulate or alleviate the cancer or hyperproliferative disorder to be treated.

The term "carrier" may be incorporated or combined with a growth factor or growth factor fragment as used herein to add or expose a growth factor or portion of the growth factor to the immune system of a human or animal and endogenous growth factor (s). Means a structure that makes the growth factor-carrier composition antigenic). The term “carrier” further encompasses a method of delivery in which a growth factor or growth factor fragment composition can be transported to a desired site by a delivery mechanism. One example of such a delivery system is the use of colloidal metals such as colloidal gold (PCT / US94 / 03177, filed March 18, 1994).

In addition, the term “carrier” further includes vaccine delivery mechanisms known to those skilled in the art, including but not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), and other adjuvants. It is also to be understood that the antigenic growth factor-containing compositions of the present invention may further comprise adjuvants, preservatives, diluents, emulsifiers, stabilizers and other ingredients used in the prior art vaccines. Adjuvant systems known in the art can be used in the compositions of the present invention. These adjuvants include incomplete Freund's adjuvant, complete Freund's adjuvant, polydisperse β- (1,4) bound acetylated mannan ("Acemannan"), TITERMAX ^R (polyoxyethylene-polyoxypropylene copolymer adjuvant from CytRx Corporation ), Modified lipid adjuvant from Chiron Corporation, saponin induction adjuvant from Cambridge Biotech, killed Bordetella pertussis , lipopolysaccharides of gram-negative bacteria (LPS), large polymer anions such as dextran sulfate and alum, aluminum hydroxide Or inorganic gels such as aluminum phosphate.

Carrier proteins that can be used in the antigenic growth factor-containing compositions of the invention include maltose binding protein "MBP"; Bovine serum albumin "BSA"; Keyhole limpet hemocyanin "KLH"; Ovalbumin; Flagellin; Tyroglobulin; Serum albumin of any species; Gamma globulin of any species; Cells of prehistoric synchrony; Genetic syngeneic cells with the Ia antigen; And polymers of D- and / or L-amino acids.

In addition, the term “effective amount” refers to the amount of growth factor that, when administered to a human or animal, elicits an immune response, prevents cancer, eliminates cancer, or inhibits the spread and proliferation of cancer. Effective amounts are readily determined by those skilled in the art according to the routine procedures described below.

For example, the immunogen growth factor compositions of the present invention may be administered parenterally or orally in the range of approximately 1.0 μg to 1.0 mg per patient, but this range is not limited. The actual amount of growth factor composition needed to elicit an immune response will vary from patient to patient depending on the immunogenicity of the growth factor composition administered, the condition to be treated and the immune response of the individual. As a result, the specific amount to be administered to an individual will be determined by routine experimentation and is based on the training and experience of those skilled in the art.

Growth factor-containing compositions of the invention are used to produce antibodies against a portion of growth factors that have become immunogens due to their addition to a carrier. Compositions comprising growth factors in combination with a carrier can be administered directly to a patient to elicit an immune response. In contrast, anti-growth factor antibodies can be administered to a subject to passively immunize them against growth factors to prevent the onset of cancer growth, to eliminate existing cancer or to inhibit the growth of cancer.

In one aspect, the invention includes a growth factor inserted into a carrier, such as a membrane carrier, such that a portion of the growth factor is added to the carrier surface. Growth factors are usually not immunogens because they are recognized by the immune system by themselves. However, introducing a growth factor in the carrier into the host system, for example by inserting a portion of the growth factor on the surface of the liposome, modifies the addition of the growth factor to the immune system to make it an immunogen.

Immunogen growth factor-containing liposomes can be prepared by reconstituting the liposomes in the presence of purified or partially purified growth factors. In addition, growth factor peptide fragments may be reconstituted into liposomes. The invention also includes growth factor and growth factor peptide fragments modified to increase their antigenicity. For example, antigenic residues and auxiliaries may be attached to or mixed with the growth factors. Examples of antigenic residues and auxiliaries include, but are not limited to, lipophilic muramyl dipeptide derivatives, nonionic block polymers, aluminum hydroxide or aluminum phosphate adjuvants and mixtures thereof.

The invention further includes growth factor fragments modified with hydrophobic residues, such as palmitic acid, which facilitate the insertion of the carrier into the hydrophobic lipid bilayer. Hydrophobic moieties of the invention may be fatty acids, triglycerides and phospholipids, wherein the fatty acid carbon backbone has at least 10 carbon atoms. Most preferred are lipophilic moieties having fatty acids as the carbon backbone of at least about 14 to about 24 carbon atoms. Most preferred hydrophobic moieties have a carbon backbone of at least 14 carbon atoms. Examples of hydrophobic residues include, but are not limited to, palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. The most preferred hydrophobic residue is palmitic acid.

Immunogen compositions containing growth factors, modified growth factors, and peptide fragments thereof are administered to humans or animals to induce immunity to growth factors. Immunized humans or animals produce circulating antibodies against growth factors that bind to growth factors, reducing or inactivating their ability to stimulate cancer cell proliferation.

Liposomes with growth factors inserted into the membrane and other immunogenic compositions containing growth factors are also used to produce panels of monoclonal or polyclonal antibodies specific for growth factors. Antibodies are prepared by methods well known to those skilled in the art. Anti-growth factor antibodies, when administered to an individual, bind to growth factors and reduce the effective concentration of circulating growth factors. As a result, growth factor-dependent proliferation of cancer is prevented, alleviated or inhibited.

Growth factor-containing compositions and anti-growth factor antibodies are administered to a human or animal by any suitable means, preferably by injection. For example, growth factors reconstituted in liposomes are administered subcutaneously. Whether produced internally or from an external source, circulating anti-growth factor antibodies bind to growth factors and reduce or inactivate their ability to stimulate cancer cell proliferation.

Liposomes that can be used in the compositions of the present invention include those known to those skilled in the art. Any standard lipid useful for preparing liposomes can be used. Standard bilayer and multilayer liposomes can be used to prepare the compositions of the present invention. Although methods for preparing liposomes known to those skilled in the art can be used, the most preferred liposomes are Alving et al., Infect. Immun. 60: 2438-2444, prepared according to the method of 1992. Liposomes may optionally contain adjuvants. Preferred adjuvants are detoxified lipid A, such as monophosphoryl or diphosphoryl lipid A.

If the vesicles are liposomes, growth factors generally have a hydrophobic tail inserted into the liposome membrane when formed. In addition, growth factors can be modified to contain hydrophobic tails that allow growth factors to be incorporated into liposomes. For example, growth factor genes are fused to oligonucleotide sequences encoding hydrophobic tails. The modified gene is inserted and expressed in an expression system using methods known in the art to produce growth factor fusion proteins with hydrophobic tails. In contrast, growth factors are exposed to the surface of liposomes already formed by chemical attachment or electrical insertion.

If the vesicle is a baculovirus-derived vesicle, the recombinant growth factor is expressed in the membrane of the insect cell as a natural result of processing by the infected insect host cell. Like the liposome embodiments described above, growth factors may be modified such that the recombinant expressed protein contains a hydrophobic moiety to facilitate insertion into the vesicle membrane.

Growth factor antibodies

The antibodies provided herein are monoclonal or polyclonal with binding specificities for growth factors such as bFGF and VEGF and antigenic peptides and fragments thereof. Preferred antigenic fragments include peptides having the amino acid sequence set forth above in SEQ ID NOs: 1-9. Most preferred antibodies are monoclonal antibodies because of their high specificity for antigen. Antibodies are antigen specific and have minimal or no cross-reactivity with other growth factor proteins or peptides.

Monoclonal antibodies of the invention are prepared by immunizing an animal, such as a mouse or rabbit, with a total growth factor (eg bFGF) or an immunogen fragment thereof (eg heparin binding domain peptide of bFGF). Splenocytes are harvested from immunized animals and hybridomas are produced by fusing sensitized splenocytes with myeloma cell lines such as rat SP2 / O myeloma cells (ATCC, Manassas, VA). The cells are induced to fuse by the addition of polyethylene glycol. Hybridomas are chemically selected by plating the cells in a selection medium containing hypoxanthine, aminopterin and thymidine (HAT).

Hybridomas are then screened for their ability to produce monoclonal antibodies against specific growth factors or related proteins and fragments. Growth factors and related proteins and peptides used for selection purposes are obtained from analytical specimens. Alternatively, the growth factor of interest may include recombinant peptides prepared according to methods known to those skilled in the art. Hybridomas that produce antibodies that bind growth factors or protein fragments thereof are cloned, expanded and cryopreserved for further production. Preferred hybridomas produce monoclonal antibodies with IgG isotypes, more preferably IgG1 isotypes.

Polyclonal antibodies are prepared by immunizing an animal, such as a mouse or rabbit, with the aforementioned growth factors or protein fragments thereof. Blood serum is then collected from the animals and antibodies in the serum are selected for binding reactivity to antigens that are reactive to growth factors or protein fragments, preferably the monoclonal antibodies described above.

Peptide or Protein Fragment

Growth factors, peptides or fragments thereof containing an immunogen region can be produced from the above-described proteins and tested for immunogen activity using techniques and methods known to those skilled in the art. For example, full length recombinant bFGF (rbFGF) can be produced using a baculovirus gene expression system. Full length proteins can be cleaved or digested into individual domains using a variety of methods, such as, for example, the method described by Enjyoji et al. (Biochemistry 34: 5725-5735 (1995)). According to the method of Enjyoji et al., RbFGF is treated with a digestive enzyme, human neutrophil elastase, and the digest is purified using a heparin column. Human neutrophil elastase cleaves bFGF into several fragments: one contains the heparin binding domain and the other contains the receptor binding domain. To produce additional fragments, the protein is preferably treated with the decomposition compound, hydroxylamine, according to the method of Balian et al. (Biochemistry 11: 3798-3806 (1972)), and the degradation product is purified using a heparin column. do.

Alternatively, fragments are prepared by digesting an entire protein or immunological fragment thereof showing immunogenic activity to remove one amino acid at a time. Each shorter fragment is then tested for immunogen activity. Likewise, fragments of various lengths can be synthesized and tested for immunogen activity. By extending or shortening the length of the fragments, one skilled in the art can determine the exact number, identification and sequence of amino acids in the protein required for immunogen activity using routine digestion, synthesis and screening procedures known to those skilled in the art. Can be.

Desired peptides and peptide fragments can also be obtained from commercial sources such as Infinity Biotech Research and Resources (Upland, PA) or Research Genetics (Huntsville, AL).

Recognizing that the progressive growth and development of primary tumors and metastatic lesions depends on angiogenesis, considerable effort has focused on the potential therapeutic manipulation of the angiogenic compartment of the tumor. In this regard, principles have been found aimed at specific immune responses to appropriate epitopes on FGF molecules for endogenous regulation of tumor growth. Peptides derived from the functional domain of FGF are selected for their ability to competitively inhibit FGF stimulated proliferation of endothelial cells. Peptides covalently bound to transparent vesicles containing lipid A as an adjuvant that block endothelial cell proliferation but do not block tumor cell proliferation in vitro as a target antigen and used to vaccinate mice, an angiogenesis stimulant Elicit specific immune response to FGF. Using this liposome technology, the inventors of the present invention found that vaccination with heparin binding domains, rather than the receptor binding domains of mice, results in inhibition of angiogenesis in vivo and the growth and development of tumors in two mice in an experimental metastasis model. Has been shown to significantly reduce. Thus, reversal of the angiogenic phenotype of malignant cancer can be achieved through the inhibition of positive regulators of vascular development.

Molecular characterization of basic and acidic fibroblast growth factor (FGF) confirms the presence of two classes of closely related angiogenic factors. Acidic and basic FGFs (hereafter aFGF and bFGF) have been identified in several molecular forms that are proteolytic processing products at homologous sites. FGF is involved in many physiological functions such as regeneration, growth and development. The identification of specific functional domains in the primary structure of bFGF was tested by Baird et al. (Proc. Natl. Acad. Sci. USA 85 (1988)), incorporated herein by reference.

Basic fibroblast growth factor (bFGF) is an important stimulant of angiogenesis involved in tumor progression. As described by Examples 1-15, two synthetic peptides, one derived from the heparin-binding domain and the other from the receptor binding domain of bFGF, inhibit in vitro bFGF stimulated HUVEC proliferation. C57BL / 6J mice are each vaccinated with synthetic peptides covalently conjugated to liposome vesicles containing lipid A. Serum analysis showed antibody response in mice immunized with heparin-binding domain peptide, but no antibody response in mice vaccinated with control liposomes or receptor binding domain peptides conjugated to liposomes. To determine if vaccination affects the bFGF stimulated response in vivo, gelatin sponges containing bFGF are transplanted into the liver of vaccinated mice. Histological analysis of the sponges showed no cell infiltration and angiogenesis of the sponges in mice vaccinated with heparin-binding domain peptides. Thus, growth factor vaccines comprising particularly immunogenic heparin-binding domain peptides of bFGF can be used to stimulate immune responses that correlate with inhibition of bFGF stimulated in vivo responses.

Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and is involved in processes involving angiogenesis and vascular permeability. VEGF is a 40-45K homodimer and in a limited manner has an amino acid sequence similar to that of platelet-induced growth factor (Soker et al. Cell, 92: 735-745 (1998), incorporated herein by reference in its entirety). As described in Example 14, previously unidentified immunogen fragments of VEGF are administered to the host to elicit an active immune response, inhibiting tumor growth and removing cancer.

In accordance with the novel findings described herein, growth factors such as bFGF peptides and / or VEGF peptides can be used in compositions suitable for treating angiogenic-related diseases, particularly hyperproliferative disorders such as cancer.

The active fragment is preferably a fragment containing an immunogen portion of the total growth factor or an immunogen peptide thereof. Preferably, immunogen proteins or fragments useful in the present invention include bFGF, VEGF and immunogen fragments thereof. More preferably, the immunogen fragment comprises a peptide having the amino acids set forth in SEQ ID NOs: 1-9. Most preferably, the immunogen peptide comprises a fragment having SEQ ID NO: 1. Immunogen fragments useful in the present invention include:

Peptide A:

YCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEEGVVSIKGV (SEQ ID NO: 1)

Peptide B:

SNNYNTYRSRKYSSWYVALKR (SEQ ID NO: 2)

Peptide C:

CRTKPEKCDKPRR (SEQ ID NO: 3)

Peptide D:

CECRPKKDRTKPEKCDKPRR (SEQ ID NO: 4)

Peptide E:

APTTEGEQKSHEVIKFMDVYC (SEQ ID NO: 5)

Peptide F:

CERRKHLFVQTCKCSCKNTDSRCKARQLENERTCRCDKPRR (SEQ ID NO: 6)

Peptide G:

CNDEGLESVPTEESNITMQIMRIKPH (SEQ ID NO: 7)

Peptide H:

CNDEGLESVPTEE (SEQ ID NO: 8)

Peptide I:

CEESNITMQIMRIKPH (SEQ ID NO: 9)

Peptide J:

YCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGV (SEQ ID NO: 10)

Formulation

Natural or synthetic proteins, peptides or protein fragments containing all or an active portion of an immunogenic protein or peptide can be prepared in a physiologically acceptable formulation, such as in a pharmaceutically acceptable carrier, using known techniques. have. For example, a protein, peptide or protein fragment can be combined with a pharmaceutically acceptable excipient to form a therapeutic composition.

Alternatively, genes for proteins, peptides or protein fragments, containing all or an active portion of an immunogen peptide, can be delivered to a vector for continuous administration using gene therapy techniques. The vector can be administered in a vehicle having specificity for a target site such as a tumor.

The composition of the present invention may be administered in the form of a solid, liquid or aerosol. Examples of solid compositions include pills, creams, and injectable dosage units. Pills can be administered orally. Therapeutic creams may be administered topically. Injectable dosage units can be administered locally, for example at the site of the tumor, or can be injected, eg subcutaneously, for systemic release of the therapeutic composition. Examples of liquid compositions include intramuscular, subcutaneous, intravenous, intraarterial injection formulations and formulations for topical and intraocular administration. Examples of aerosol formulations include inhalation formulations for administration to the lungs.

The composition can be administered by standard routes of administration. In general, the compositions can be administered by topical, oral, rectal, nasal or parenteral (eg, intravenous, subcutaneous or intramuscular) routes. In addition, the composition may be introduced into a sustained release matrix, such as a biodegradable polymer, wherein the polymer is injected around a desired delivery site, for example a tumor site. The method includes administration of a single dose, repeated administration of the dose at predetermined intervals, and sustained administration for a predetermined time.

As used herein, a sustained release matrix is a matrix made of a substance, typically a polymer, that is degradable by enzymes or acid / base hydrolysis or dissolution. Once inserted into the body, the matrix acts by enzymes and body fluids. The sustained release matrix is preferably liposome, polylactide (polylactide acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (copolymer of lactic acid and glycolic acid), polyanhydride, poly (Ortho) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, phenylalanine, tyrosine, amino acids such as isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone It is selected as biocompatible materials such as don and silicone. Preferred biodegradable matrices are one of polylactide, polyglycolide or polylactide co-glycolide (a copolymer of lactic acid and glycolic acid).

The dosage of the composition will depend on other clinical factors, such as the condition to be treated, the particular composition used and weight, and the condition and route of administration of the patient.

The composition may be administered in conjunction with other compositions and procedures for treating the disease. For example, unwanted cell proliferation can typically be treated with surgery, radiation or chemotherapy with administration of the composition, and additional doses of the composition will be subsequently administered to the patient to stabilize and inhibit the growth of the remaining unwanted cell proliferation. Can be.

Diseases and Symptoms to Treat

The methods and compositions described herein include hemangiomas, solid tumors, leukemias, metastasis, capillary dilemma, psoriasis, scleroderma, purulent granulomas, myocardial angiogenesis, plaque angiogenesis, corneal disease, skin redness, neovascular glaucoma, diabetic Abnormal or unwanted, including but not limited to retinopathy, posterior capsular fibrosis, arthritis, diabetic angiogenesis, macular degeneration, wound fusion, peptic ulcer, fracture, keloid, angiogenesis, hematopoiesis, ovulation, menstruation and placental formation Useful for treating diseases and processes in humans and animals that are mediated by cell proliferation, particularly by abnormal or unwanted endothelial cell proliferation. The methods and compositions are particularly useful for treating angiogenesis-related disorders and diseases by inhibiting angiogenesis.

The methods and compositions described herein are particularly useful for the treatment of cancer, arthritis, macular degeneration and diabetic retinopathy. Administration of the composition to humans or animals with preangiogenic metastasized tumors is useful for preventing the growth or expansion of such tumors.

The compositions and methods of the present invention are further illustrated by the following non-limiting examples, which do not in any way limit the scope thereof. Conversely, after reading the description herein, there may be various other aspects, modifications, and equivalents thereof that can be described to those skilled in the art without departing from the spirit of the invention and / or the scope of the appended claims. It should be clearly understood.

Example 1

Construction of Growth Factor-Containing Recombinant Baculovirus

Baculovirus expression vectors are described in Webb, NR et al. (1989) Proc. Natl. Acad. Sci. It is constructed as described in USA 86, 7731-7735. Recombinant baculoviruses containing cDNA encoding full-length growth factors under the transcriptional control of the polyhedron promoter are characterized by calcium phosphate precipitation from recombinant pAc-growth factor DNA and wild-type Autographa californica nuclear polyhedronogenic virus (AcMNPV) DNA. Produced by co-transfection.

Example 2

Expression and Purification of Growth Factors

The occlusal-negative virus of Example 1 is plaque-purified and propagated in Spodopterafrugiperda 9 (Sf-9) cells. Infected Sf-9 cells were isolated from Webb, NR et al. (1989) Proc. Natl. Acad. Sci. Proliferate into monolayers or suspensions as described in USA 86, 7731-7735. In brief, Sf9 cells are described in Summers, MD and Smith, (1987) A Manual of Methods for Baculovirus Expression Vectors and Insect Cell Culture Procedures (Texas Agricultural Experiment Station, College Station TX), Bull. As described in 1555, it is cultured in TNMFH medium at 27 ° C. supplemented with 10% v / v heat-inactivated fetal bovine serum. Extraction and purification of recombinantly expressed growth factors is performed by standard methods.

Example 3

Preparation of Growth Factor-Containing Liposomes

Purified growth factors are electroinserted into liposomes (Mouneime, Y., et al., 1990, Biochem., Incorporated herein by reference) or reconstituted by standard methods known in the art. Incorporation of growth factors or fragments thereof into liposomes may include:

(a) Rehydrating lyophilized liposomes of known lipid components in the presence of aqueous growth factor (or growth factor fragments) aqueous solution so that growth factors are contained in the lumen of liposomes or trapped in the aqueous layer between the lipid membranes of the multilayer plate vesicles. Or preparing multi-lamellar liposomes;

(b) electroinsertion of growth factors or fragments thereof into reconstituted liposomes (growth factors are present between the lumen or lipid membrane);

(c) liposome reconstitutions prepared with a composition known as lyophilized fragments of growth factors, each fragment containing a hydrophobic tail inserted directly into the liposome lipid bilayer such that the peptide fragments are exposed to the outer surface of the liposomes;

(d) conjugation of the active reactant to the liposome surface; And

(e) a method of reconstitution or formulation of an active reactant which results in an immune response (humidity or cellular) against a synthetic peptide that mimics the composition and activity of a growth factor, fragment thereof or growth factor.

Example 4

Preparation of Growth Factor-Containing Baculovirus Vesicles

Growth factor-containing vesicles are produced as follows. Insect-derived vesicles containing recombinant growth factors in the membrane are obtained using baculovirus-infected insect cells. In more detail, Spodoptera frugiperda IPLB-Sf21-AE clone isolate 9 (named Sf9) is cultured in insect cells and is incorporated herein by reference, Webb et al., Proc. Natl. Acad. Sci., 86: 7731-35 (1989) and US Pat. Nos. 4,745,051 and 4,879,236 (both Smith et al., Both), recombinant baculos containing cDNA encoding full-length growth factors Infect with virus. Approximately 0.8 × 10 ⁶ Sf9 cells are seeded in a 1 liter Spinner flask containing Excell medium (JRH Scientific, Woodland, CA 95695). Cells are incubated in 27 ° C., 50% O ₂ atmosphere. Once the cells reach a density of 3.5 to 4.0 million cells / ml, baculovirus containing the recombinant growth factor is added to the medium at a multiplicity of 400-600 viruses / cells. Vesicle production begins about 24 hours after baculovirus infection. Peak vesicle formation is achieved approximately 72 hours after initial baculovirus infection. The flask contents are collected and centrifuged at approximately 1200 rpm to remove cells and debris. The supernatant containing the vesicles is collected and centrifuged in 50% Percoll containing 0.1 M sodium bicarbonate pH 8.3 using a fixed angle rotor for 30 minutes at 20,000 RPM. Double bands are collected under the interface between Percoll and cell culture medium and the suspension is centrifuged for 30 minutes with a swing-bucket rotor at 20,000 RPM. Two bands can be observed at the top and bottom of the gradient with a density of 1.05 g / ml for vesicles and 1.06 g / ml for baculovirus particles. Vesicles can be used for immunization with growth factors added to their outer surfaces. The vesicles are washed three times with 0.1 M sodium bicarbonate pH 8.3 using 20,000 RPM centrifugation for 20 minutes and resuspended in the same buffer.

Example 5

Immunization with Growth Factor-Containing Compositions

Immunogen growth factor-containing compositions, including liposomes or baculovirus-induced vesicles, are injected into humans or animals at a dose of 1-5000 μg / kg body weight. Antibody titers to growth factors when present or developing specifically for each growth factor are determined by ELISA or other serology techniques using recombinant proteins and horse-radish peroxidase-conjugated goat anti-human or animal immunoglobulins. Sandwich ELISA) or biological activity assays (eg, neutralization or growth factor assays or competition assays of natural or synthetic cytokines). Booster injections are administered as needed to achieve levels of protective antibodies sufficient to reduce or neutralize the growth factor activity in vivo. Neutralization titers and appropriate antibody isotypes are determined in experimental animals challenged with appropriate cancer cells.

Example 6

Preparation and Isolation of Anti-Growth Factor Antibodies

Individuals with a strong immune system are immunized as described in Example 5. After high titers of anti-growth factor antibodies are achieved, IgG fragments are separated from the blood and used to passively immunize the subjects as described in Example 7 below.

Example 7

Passive immunization

Anti-growth factor antibodies isolated from passively immunized species are administered by intravenous injection at a dosage level of approximately 0.5-50 mg per kg body weight. Dosage and frequency of administration were determined for experimental animals challenged with different tumor types and adjusted according to the particular type of tumor and the particular subject to be treated. Important considerations are the aggressiveness of the tumor, the propensity of metastatic spread, the metastatic target organ, the tissue accessibility of the target organ angiogenesis / antibody, and the stage of tumor development. It may be true that standard methods that would normally be protective may be determined, but it may also be true that effective therapies can only be achieved with differentiated criteria based on the tumor type.

Example 8

Preparation of Peptide Conjugated Liposomes as Anti-bFGF Vaccine

The following methods and protocols are used for the production of anti-growth factor vaccines, in particular anti-bFGF vaccines.

Selection of Peptides

Peptides selected for use in the present invention may comprise whole growth factor proteins or immunogen fragments thereof. In the case of this example, the immunogen peptide is selected from the bFGF amino acid sequence.

Introduction of FGF Peptides into Liposomes

Dimyristoyl-glycero-3- [phospho-rac- (1-glycerol)] (DMPG), Dimyristoyl-glycero-3-phosphocholine, containing 5 mg / ml of lipid A ( Liposomes consisting of DMPC), cholesterol (Avanti Polar Lipids, Alabaster, AL), pyridyl dithiocholesterol (PDS-Chol) and lipid A (List Biological Laboratories, Inc. Campbell, CA) Prepared in molar ratio. The lipids are mixed in a round bottom flask and the solvent is removed by rotary evaporation. Multilayer plate vesicles are prepared by hydrating dry lipids with 6.4 ml of sterile water (BioWhittaker, Walkersville, MD) containing 2 mg of heparin binding domain peptide or receptor binding domain peptide of FGF-2. The liposome formulations are incubated overnight at room temperature (RT) with gentle orbital shaking, lyophilized and resuspended in 2.135 mL of 0.1 M citrate phosphate buffer pH 5.6. 200 microliters of each peptide are added to each liposome preparation and then incubated for 16 hours at RT on an orbital shaker. Liposomes are centrifuged at 15,000 rpm at 4 ° C. for 30 minutes. For injection, liposomes are resuspended in a final volume of 5 ml of non-pyrogenic Mg ²⁺ and Ca ²⁺ free PBS. Quantification of the peptide bound to liposomes is performed by amino acid analysis (MA BioServices, Rockville, MD).

group

-8 full groups initially immunized

3 groups immunized with a composition comprising liposomes conjugated to peptides

3 groups immunized with a composition comprising liposomes in which the peptide is passively encapsulated

-Liposome 1 group with buffer only, using lipid instead of conjugated liposomes

Liposome 1 group with buffer only, using lipid instead of passive encapsulation

All contain lipid A ⓒ 22 μg lipid A / micromolar phospholipids.

Immunization & Treatment of Mice

The animal model used for this example consisted of Balb / CyJ mice. Five mice were used in each of the eight groups.

C57BL / 6J male mice, aged 5-7 weeks, without specific toxins are purchased from The Jackson Laboratory (Bar Harbor, ME). Mice are placed in a facility free of specific pathogens. When conducting research, researchers follow the Guide for Laboratory Animals and Care of the Institute of Laboratory Animal Resources, the National Academy of Sciences, and the National Research Council. Mouse populations are treated three times (0.1 mL, intramuscularly) with liposomes containing heparin binding domain peptide (L (HBD)) or receptor binding domain peptide (L (RBD)) at two week intervals. Each liposome injection delivers 1 micromole of phospholipid, 200 μg lipid A and 30 μg peptide. Control groups include liposomes without conjugate peptides (L (LA)) and PBS. Thirteen days after the last injection, the tail veins of the mice are bleeding and serum is collected for analysis of the antibodies. The next day mice are challenged intravenously with tumor cells or with a sponge (liver insert) injected with FGF-2 or B16BL6.

Liposome

MLV (10 mol% PDS-CHOL) containing DMPC: DMPG: CHOL: PDS-CHOL: MLA molar ratio of 9: 1: 7.4: 2: 0.02 for conjugated pentides

MLV containing DMPC: DMPG: CHOL: MLA molar ratio of 9: 1: 7.5: 0.02 for passive encapsulation

Dosage

Target dosages include approximately 10-50 μg peptide and 100 μg MLA. Sufficient amount for 3 injections per mouse during the whole procedure.

material

100 ml round bottom flask, 24/40 joint, depyrogenated, 35 ml Oakridge tube. All materials were properly sterilized and autoclaved.

Distribute lipids into all 8 groups ‡ (per group)

Geology [Stock] mM μmol Ml stock Molar ratio DMPC 90 204 2.27 9 DMPG 10 23 2.3 One CHOL 75 170 2.27 7.4 PDS-CHOL 20 44 2.2 2 (20 mM = 10.26 mg PDS-Chol / mL)

MLA is added at 22 μg / μmol; Lot # 4013B from List Biologicals, Inc.

• 100 μg MLA assumption per injection, sufficient for approximately 50 injections;

Approximately 50% peptide encapsulation assumption using 1.5 mg total peptide and 10 μg per injection, sufficient for approximately 50 injections;

Dissolve and pool # 1 mg / vial MLA in approximately 1 ml / vial 1: 1 chloroform: methanol.

Preparation of Pyridyldithio-cholesterol (PDS-Chol) for Surface Conjugation of Cysteine-terminated Peptides to Liposomes

To make the relatively non-immunogenic peptides more immunogenic, surface conjugation of cysteine-terminated peptides derived from the VEGF amino acid sequence to liposomes is performed. PDS-Chol is a thiol-reactive cholesterol analog that binds to free thiols. The peptide has an amino- or carboxyl-terminal thiol that will bind PDS-Chol in an aqueous liposome environment. Peptides are conjugated to multilayer plate vesicles (MLV; liposomes) both inside and outside of the MLV to promote depo or slow release effects of antigen presence.

The method used includes modifications of Carlsson, et al.

1 day

1. Dissolve 1.772 g (44 mmol) of thiocholesterol (Aldrich Chem., # 13,611-5) in 2.6 mL of chloroform. A 13 × 100 mm glass screw-cap tube is used and the composition is kept under nitrogen. The composition is almost completely dissolved in the mixing line and slightly milky.

2. 1.9'g (9.0 mmol) of 2 ', 2'dipyridyldisulfide (PDS) (Aldrithiol-2; Aldrich Chem., Milwaukee, Wisconsin) was placed in a 25 ml vaccine vial and 100: 1 (v: v) Chloroform: 5.6 ml glacial acetic acid is added. Add a stir bar and mix the composition until dissolved (about 2 minutes).

3. Add thiocholesterol to the vigorously mixed PDS at a rate of approximately 30 drops per minute. Yellow appears.

4. Immediately after addition of thiocholesterol, the vial is purged with nitrogen, sealed, covered with aluminum foil and mixed overnight at room temperature.

2 days

5. Evaporate the solvent from the vial in the hot water bath into the nitrogen stream. Dry with viscous oil. Acetic acid is identified as an odor.

6. Transfer the composition to a 250 ml round bottom flask for recrystallization using chloroform and wash the reaction vial.

7. The composition is rotary evaporated to remove chloroform to give a viscous tan oil. The nitrogen stream is reapplied to remove residual solvent.

8. The composition is recrystallized by adding 50 ml of ethanol at about 56 ° C. Vortex until dissolved. The sample was completely dissolved. If left at room temperature, an emulsion is obtained (about 20 minutes). Leave on ice for approximately 1 hour. Large white crystals are formed.

9. Place at -20 ° C for 2 days to complete crystallization. Large amounts of crystalline product appear. Large white / yellow crystals.

5 days

10. Recover by filtration and wash with ice-cold (dry ice) ethanol. Dry for approximately 4 hours to weigh. Confirm the structure by IR and NMR.

11. TLC in 9: 1: 1: 0.1, v / v of hexanes: ethyl acetate: diethyl ether: glacial acetic acid.

PDA-Chol PDS Thio-cholesterol Rf's 0.74 0.22 0.92

18 days

12. Second recrystallization with 100 mL of ethanol at 63 ° C. Leave at room temperature for approximately 2 hours and then at -20 ° C for 2 days.

TLC RF

Solvent system: Hexane: ethyl acetate: diethyl ether: glacial acetic acid, 9: 1: 1: 0.1, v: v

PDS-CHOL PDS Thio-cholesterol TLC 0.74 0.22 0.92 TLC 0.63 0.14 0.97

Previous synthesis

Assay plate 0.84 0.31 0.97 1 mm thickness 0.55 n / a n / a Preparative TLC Purity Check TLC 0.63 0.18 Assay plate

Stock solution: prepared from 512.77 to 20 mM = 10.26 mg / ml chloroform

Weight vs. absorbance

The analysis is based on the DTT-release of 2-thiopyridone, which strongly absorbs at 343 nm. The extinction coefficient is 7.0 / mM at 343 nm.

1. Weigh three individual samples of PDS-cholesterol in a 13 × 100 tube.

2. 2.5 mM (1.282 mg / ml) stock in ethanol of PDS-Chol and 2-PDS (2-Aldrithiol-2 ^™ ; Aldrich).

3. Dilute PDS-chol in ethanol and 2-PDS in 0.1 M sodium bicarbonate to 0.125 mM. Molecular Weight of PDS-Chol = 512.77.

4. Prepare 50 mM dithiothreitol (7.72 mg / ml) in 0.1 M sodium bicarbonate.

result

Assays using functional group-specific sprays:

Sterol spray: ferric chloride spray: specific for sterol: for the positive, 2 for the PDS-guess PDS- cholesterol and cholesterol thio voice

Iodine vapor : shows 90-95% purity (measured)

Surface Bonding of Peptides: Methods (Use Biological Safety Cabinets for Operation)

1 day

1. Dispense the lipids in the following order in the pyrogen-free 1-ml round bottom flask; Lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol.

2. Rotate evaporate to remove bulk solvent. The bath is 42 ° C. Flask rotation was set to 160 rpm. Start at about 22 mmHg vacuum until the bulk solvent evaporates, then raise the vacuum to 25 mmHg and run for 5 minutes. Nitrogen is blown into the flask and plugged until the flask is ready for drying.

3. Cover the flask inlet with filter paper and dry under vacuum for 3 hours.

4. Deoxygenate the sterile water by nitrogen bubbling through a sterile 5 ml pipette capped for 20 minutes while drying the lipid. Also 10 ml of 10 mM acetic acid are prepared and the filter is sterilized. Stock HOAc is 17.5 N. 20 microliters of stock HOAc is added to 35 ml of water to give 10 mM. pH is 3.4. Deoxygenate with nitrogen bubbling and sterilize the 0.22 micron syringe filter.

5. Completely dissolve the peptides to 5 mg / ml with 10 mM sterile acetic acid.

Peptide information is listed in the table below:

Peptide Experiment # Produce # Physical state BFGF Research Genetics, Huntsville, Alabama Fluffy White Powder

Solubility

White powder is completely dissolved; Actually immediately.

6. Add 0.2 ml (1 mg) of 5 mg / ml peptide solution to 6.4 ml of deoxygenated DIW and mix gently. This is a peptide hydration solution.

7. Add peptide hydration solution to the dried lipids. The flask is purged with nitrogen, glass-stopped and sealed with parafilm.

8. Sonicate the bath to resuspend lipids. A milky suspension was produced. Vortex briefly.

9. Place on an orbital shaker, cover with foil and set to 3.25 to spin overnight at room temperature.

2 days

10. Transfer the preparation to a sterile 20 ml vaccine vial and lyophilize for 3 days.

3 days

11. Prepare 0.1 M citrate / phosphate buffer, pH 5.6 (C / P buffer): Dissolve 1.921 g of citric acid in 100 mL DIW (“A”); 5.37 g of dibasic & odium phosphate-7-hydrate are dissolved in 100 ml of DIW (“B”); 21 ml "A" and 29 ml "B" are mixed and 50 ml DIW is added. Deoxygenate with nitrogen bubbling and sterilize the filter.

12. Pre-form liposomes by adding 2.135 mL of sterile C / P buffer, purging the vial with nitrogen and vortexing vigorously for 1-2 minutes. The suspension is relatively homogeneous. Lyophilisation may be a limiting factor due to the large ratio of fluid to vial capacity.

13. Dissolve each peptide completely to 10 mg / ml in sterile, degassed 10 mM acetic acid. The powder dissolves immediately and the solid dissolves completely in about 1 minute by vortexing.

14. Add 0.2 ml (2 mg) of 10 mg / ml peptide / HOAc solution to the preformed vesicles, purge the vial with nitrogen, seal and then cover with foil.

15. Mix overnight at room temperature with an orbital shaker set to 6.

4 days

16. First Spin: Continuous wash the vials with 2.5 mL total sterile PBS, two fractions of 1 mL PBS followed by 0.5 mL PBS for lipids. A 1 ml plastic pipette is best to use as a delivery tool. The total volume of the preparation is 5 ml. When measuring the volume of lipid pellets it is measured to 1.5 ml. Use sterile PBS and autoclaving Qakridge tubes.

17. With SS-34 rotor, centrifuge at 15,000 rpm at 40 ° C. for 30 minutes at Sorvall high speed.

18. Carefully pipette and freeze the supernatant. Mark the tube as the first spin. 5 ml total volume; Lipid volume 1.5 ml; Thus about 3.5 ml are aqueous and should be used for calculations, where they use each peptide stock as standard in BCA analysis.

19. Second Spin: Add about 5 ml of PBS to the pellet and vortex to completely resuspend the pellet. 30 ml of PBS total is added and inverted to mix.

20. Centrifuge at 15,000 rpm at 4 ° C for 30 minutes at high speed Sorvall with SS-34 rotor.

21. The supernatant is cryopreserved according to a 50 ml tube labeled Second Spin. Control pellets are looser in both groups. Peptide pellets are hard and the supernatant is transparent.

22. Add about 1 ml of PBS to the pellet and carefully resuspend the pellet in a homogeneous suspension. Transfer to 5 ml sterile snap-cap tubes. Sufficient PBS is used to prepare a total 5 ml suspension.

23. phosphorus analysis; Introduction assay by BCA (each peptide used as its own standard). 10 mg / ml frozen peptide stock is used.

Injection

Optimum = 100 μl i.p. containing 100 μg lipid A and 10 μg peptide.

Simple Encapsulation of Peptides: 1 Day

1. Dispense the lipids in the pyrogen-free 100 ml round bottom flask in the following order: lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol.

2. Rotate evaporate to remove bulk solvent. Start at about 22 mmHg vacuum and raise to 25 mmHg until the bulk solvent evaporates and run for 5 minutes.

3. Dry under vacuum for 2 hours.

4. Hydrate lipids as follows; 4.5 ml of sterile water is added to the lipid and the bath is sonicated to suspend the lipid. A relatively homogeneous suspension is produced. Allow to stand at room temperature for 2 hours.

5. Transfer to a 10 ml vaccine vial. The rotary flask is washed continuously with 1 ml fractions of sterile water. The vial is sealed with sterile butyl rubber vaccine vial septum and parafilm.

6. Freeze the vial for 30 minutes on dry ice.

7. Insert a # 20 sterile hypodermic needle through the septum to allow air flow for lyophilization.

8. Place the vial into the freeze dryer. Two days will be enough to lyophilize the lipid.

2 days

9. Prepare concentrated peptide solution (10 mg / ml) in 10 mM HOAc. 0.5 ml is required for 5 mg total peptide. Once dissolved, the peptide solution is diluted with 2 ml of sterile, room temperature PBS.

10. Next, 4.5 ml of the peptide solution is added to the lyophilized lipid and the vial is vigorously stirred for 2 minutes. A final concentration of about 100 mM is provided for phospholipids.

11. Place the vial at 4 ° C. for about 24 hours to allow encapsulation.

3 days

12. Transfer lipids to 35 ml sterile Oakridge tubes for centrifugation. Use a 1 ml pipette as the delivery tool. Two fractions of 1 ml cold PBS are used to wash the vial. The total volume of the preparation is 7 ml. Pellet volume is measured at about 1.5 ml.

13. Centrifuge at 15,000 rpm at 4 ° C for 30 minutes at high speed Sorvall with SS-34 rotor.

14. Pipette the supernatant and cryopreserve as the first spin.

15. Add about 5 ml of PBS to the pellet and vortex to resuspend the pellet homogeneously. PBS is added up to a total of 30 ml and centrifuged as above.

16. Resuspend the pellet in PBS and transfer to 5 ml snap-cap tubes up to 5 ml total.

17. Analyze phosphorus and peptide encapsulation.

Example 9

In vivo studies to explain the efficacy of bFGF heparin binding domain peptide vaccination

Two peptides from the functional domain of the bFGF molecule were analyzed for their ability to block bFGF stimulated endothelial cell proliferation in vivo. Peptide A is a 44 amino acid peptide corresponding to the heparin-binding domain of bFGF having the sequence YCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEEGVVSIKGV (SEQ ID NO: 1).

Peptide B is a 21 amino acid peptide corresponding to a receptor binding domain having the sequence SNNYNTYRSRKYSSWYVALKR (SEQ ID NO: 2).

Peptides A and B were synthesized by Infinity Technology, Inc. (Upland PA) and hydrated to 10 mg / ml with 10 mM acetic acid for use in proliferation assays and liposome formulations.

Both peptides A and B are introduced into liposomes containing lipid A and used respectively in the vaccination protocol. Peptides are conjugated to liposomes according to the methods and protocols described above in Example 8. Mice are immunized and boosted twice, then blood is collected and pooled serum is analyzed for anti-peptide antibody titer and cross-reactivity to bFGF by ELISA.

Comparison of titer and cross-reactivity of sera from mice vaccinated with liposome heparin binding domain peptides of whole bFGF or bFGF vaccines is shown in FIG. 4 and from mice vaccinated with liposome receptor binding domain peptides of whole bFGF or bFGF vaccines. Comparison of serum titers and crossreactivity is shown in FIG. 5.

According to the vaccination protocol, mice were challenged with bFGF-impregnated sponges and 14 days after angiogenesis, the results are shown in FIG. 6.

Angioplasty Gel Foam Sponge Model

Watanabe et al. Am. As previously described by J. Path., 150: 1869-1880, gelfoam sponges (Upjohn, Kalamazoo, MI) are cut into 5 mm squares and hydrated in PBS for 24 hours at room temperature. After hydration, the sponge is blotted dry to sterile gauze and rehydrated with 80 μl of a solution containing FGF-2 [8 ng / ml] and heparin [10 U / ml]. Gelfoam sponges are incubated at 37 ° C. for 1 hour prior to transplantation of mice's livers.

Mice are anesthetized with metaphanes and small incisions are made to expose the liver. The gel foam sponge is fixed to the left mesenchymal with a cyanoacrylate adhesive and the wound is closed with a surgical suture. After 14 days of implantation, mice are decapitated and the sponges are removed from the liver and fixed in 10% buffered formalin for tissue analysis.

Metastasis Model of Laboratory Lungs

B16BL6 tumor cells in exponential phase were harvested with short trypsinization (0.25% DIFCO trypsin and 0.02% EDTA for 1 min at 37 ° C), washed and then 2.5x10 ⁵ cells / ml in Ca ²⁺ , Mg ²⁺ free PBS Resuspend. Mice are injected with a 27-gauge needle with 0.2 ml (5 × 10 ⁴ tumor cells / mouse) of cell suspension through the posterior vein. After 14 days of inoculation, mice are decapitated, the lungs are removed and fixed in formalin. Lung metastasis is counted with an anatomical microscope (Olympus, 3 ×).

The exponential LLC-LM tumor cells are harvested using a cell scraper, washed and resuspended at 1 × 10 ⁶ cells / ml in Ca ²⁺ , Mg ²⁺ free PBS. Mice are injected with a 27-gauge needle with 0.2 ml of cell suspension through the tail vein. After 17 days of inoculation, mice are decapitated and their lungs removed, weighed and fixed in 10% buffered formalin.

Histological analysis

Lung and gelfoam sponges are fixed in 10% buffered formalin. Tissues are embedded in paraffin by routine methods, cut to 6 μm and stained with hematoxylin and eosin (H & E) according to Molecular Histology Labs, Inc. (Gaithersburg, MD). Micrographs are taken using an Olympus 1 × 70 microscope.

Statistical analysis

Results are analyzed for statistical significance using a 2 Tailing Student's t-test.

To determine whether the production of antibodies to bFGF molecules affect bFGF-induced angiogenesis, gelatin sponges containing recombinant human bFGF were replaced with liposome lipid A controls, liposomes containing heparin binding domain peptides, and receptor binding domain peptides. Mice are vaccinated with containing liposomes or PBS and then transplanted into the left mesenchyme of mice. After 14 days of implantation, the sponge is removed. Vascular distribution and leukocyte infiltration are evaluated histologically. Well defined blood vessels containing erythrocyte cells were apparently removed in mice vaccinated with liposomes containing sponges removed from mice vaccinated with control preparations (liposomal lipids A and PBS) and the receptor binding domain peptides of bFGF. (Figure. Excessive cell infiltration of white blood cells and fibroblasts also occurred). Sponges removed from mice vaccinated with receptor binding domains looked similar to controls. In contrast, tissue analysis of sponges removed from mice vaccinated with the heparin binding domain peptide showed a lack of cell infiltration and angiogenesis induced by bFGF. Although red blood cells are present in histological sections of sponges taken from mice vaccinated with heparin binding domain peptides, they lack the well-defined structures surrounding them.

Delivery of HBD and RBD to Liposomal Vesicles: Induction of Antibody Responses

Peptides, HBD and RBD from both functional domains of FGF-2 are covalently conjugated to and encapsulated in liposome vesicles containing the strong adjuvant lipid A and used to treat mice every two weeks for six weeks. Each inoculation delivered 1 μmol phospholipid, 200 μg lipid A and 30 μg peptide. After treatment, mice are bled and serum is pooled for analysis of serum immunoreactivity against native FGF-2, HBD peptide and RBD peptide in ELISA format. Immune responsiveness was measured in mice treated with liposome formulations (L (HBD)) containing HBD peptides of FGF-2. This antibody responds to both HBD peptide and parent FGF-2 molecules with endpoint titers of 1: 10,000 and 1: 5,000, respectively (FIG. 20). Conversely, serum collected from mice treated with RBD peptides (L (RBD)) conjugated to liposomes or mice treated with liposomes (L (LA)) lacking conjugated peptides were expressed as receptor binding domain peptides, heparin binding domain peptides or It does not show immunoreactivity to the original FGF-2 molecule. As expected, all mice treated with liposome preparations containing lipid A adjuvant develop antibody titers to lipid A (end point dilution of serum: 1: 30,000) (data not shown).

To assess the specificity of antibodies produced in response to inoculation of L (HBD), serum from treated animals is assessed for immunoreactivity to another heparin binding molecule: Vascular Endothelial Growth Factor (VEGF). As assessed in ELISA format, serum from L (HBD) treated mice did not bind VEGF (FIG. 20).

Inhibition of B16-BL6 Induced Angiogenesis and Tumor Growth in Liver Sponge Inserts

To determine if vaccination can inhibit tumor-induced angiogenesis, gelatin sponges containing B16BL6 melanoma were added to control liposomes (liposomal lipid A), liposomes bound to heparin binding domain peptides, and receptor binding domain peptides. Mice are transplanted to the liver after vaccination with bound liposomes or PBS. After 14 days of implantation, the sponges are removed and histologically tested for vascular distribution and leukocyte infiltration. Sponges removed from mice vaccinated with liposome lipid A and PBS contain viable and proliferating B16BL6 tumor cells. In addition, angiogenesis, ie well defined blood vessels containing red blood cells, is evident in these sponges. In addition, excessive cell infiltration of leukocyte cells and fibroblasts occurred with these sponges. In contrast, tissue analysis of sponges removed from mice treated with liposomes containing FGF heparin binding domain peptide showed a decrease in FGF-induced cell infiltration and lack of angiogenesis (FIG. 3D). Red blood cells, on the other hand, are present in histological sections of sponges taken from mice treated with liposomes containing FGF heparin binding domain peptides. Similar effects are seen with sponges removed from mice vaccinated with liposomes containing receptor binding domains. Vaccination with heparin binding domain peptide blocks angiogenesis and inhibits tumor growth and development in the sponge. As found from sponges containing bFGF, there are abundant red blood cells lacking the defined structure surrounding them, with a lack of well-defined capillary structure and a marked lack of endothelial cells.

The results of these experiments are shown in FIGS. 6 and 7.

Inhibition of B16BL6-induced Angiogenesis and Tumor Growth in Liver Sponge Inserts

Gel sponge models of angiogenesis can also be used to assess angiogenesis induced by tumors. Gelatin sponges containing 5 × 10 ⁴ B16BL6 melanoma cells are transplanted into the livers of mice vaccinated with L (LA), L (HBD), L (RBD) or PBS. After 14 days of implantation, the sponge is removed and tested histologically. Sponges removed from mice treated with L (LA), L (RBD) and PBS contained B16BL6 tumor cells (FIGS. 21A-C). Angiogenesis into these sponges occurs as much as confirmed by endothelial cells that form capillary structures containing red blood cells. In contrast, sponges from mice treated with L (HBD) show no neovascularization and likewise inhibit B16BL6 tumor growth. Repeatedly, red blood cells are observed, but no blood vessel or endothelial structure surrounding them is observed.

Example 10

In vivo studies to demonstrate the efficacy of bFGF heparin binding domain peptides on lung metastasis in mice

To test the efficacy of bFGF peptides from the heparin-binding domain to inhibit cancer, groups of 10 mice are vaccinated, boosted and challenged with B16BL6 murine melanoma by the intravenous route. Peptides A and B are conjugated to liposomes according to the methods and protocols described above, in particular, described in Example 8, as described in Example 9.

At 14 days after mice were vaccinated, boosted and challenged with B16BL6 rat melanoma, the animals were decapitated and the lungs removed to count the number of melanoma tumor nodules on the surface (FIG. 1A). 1A shows (a) mice vaccinated with bFGF peptides from a heparin-binding domain conjugated to liposomes, (b) mice vaccinated with peptides from a receptor binding domain conjugated to liposomes, (c) empty liposomes Mean values of lung metastases are shown in vaccinated mice and (d) non-vaccinated mice (PBS control). 2 shows the differences in surface metastasis of lungs from the control group (liposomal lipid A, top) and lungs from the vaccinated group (heparin-binding domain peptide, bottom).

As demonstrated by the results of this experiment, and as shown in FIG. 1A, administration of a composition comprising a heparin binding domain peptide of bFGF is an effective anti-tumor composition as it significantly reduces the mean of lung metastasis.

Treatment with L (HBD) results in 96% inhibition of macroscopic B16BL6 metastasis in the lung compared to the lungs of mice treated with L (LA), L (RBD) or PBS. Mice treated with L (LA), L (RBD) or PBS have large black lesions covering the front of the lung. Tissue analysis of lung tissue from L (HBD) treated mice shows very few tumors that are less than ten cell layers thick. The control lung has a number of melanoma lesions greater than 10 cell layers thick.

Confirmation of anti-tumor efficacy is performed with a second experimental model of metastatic disease. After 17 days of intravenous challenge with LLC-LM, two mice in the control group died of advanced tumors, while the other control mice showed excessive breathing and decreased mobility. All groups are decapitated on day 17 and lungs are removed to evaluate tumors by measuring lung weight. Mice administered L (LA) had significantly larger tumors localized to the lung, whereas mice treated with L (HBD) showed a significant decrease in tumors (p <10 ⁵ ) (FIG. 1B).

Example 11

Efficacy of growth factor and liposome vaccine composition on tumor volume and size

Compositions comprising bFGF-liposomes are administered to tumor-bearing mice to monitor the efficacy of such compositions on tumor size.

Materials and methods

A. Preparation of Vaccine Including Liposomes

Liposomes used in this study are described in Verma et al. Prepared according to the method of Infect Immun 60 (6): 2438-2444 (1992). The vaccine was molar ratio of DMPC: DMPG: CHOL: LA in a rotary evaporator flask depleted of lipid, dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG), cholesterol (CHOL) and lipid A. Prepared by compounding at 7.5: 1 (mol: mol). The flask is dried under high vacuum for at least 2 hours. The lipid is hydrated with sterile water for 2 hours at room temperature. The hydration concentration used is 50 mM for phospholipids. The sample is then lyophilized. Recombinant human bFGF is encapsulated in liposomes by dissolving in PBS and combining with lyophilized lipids. Vortex the mixture vigorously to ensure resuspension of lipids and incubate at 4 ° C. for 2 days. The amount of vaccine injected is determined by the amount of protein desired per dose. In mice and possibly humans, adjuvant lipid A is generally preferred for active response. For this study, the dose of adjuvant is 200 μg per injection per μmol of total phospholipid and the antigen dose is 10 μg of bFGF per injection. The concentration of liposomes is given in total phospholipids, ie total DMPG and DMPC; The liposome (vaccine) final concentration is 10 mM phospholipid. The highest potent dose may vary and studies are underway to identify it.

B. Immunization Schedule

6-8 week old, C57BL / 6J or Balb / cByJ mice are immunized with intraperitoneal injection of liposomes containing bFGF on days 0, 14 and 21. Serum is collected on days 0, 14, 21 and 35. Serum is analyzed for anti-bFGF activity by ELISA. Control groups of mice include non-injected mice bleeding for titer and mice inoculated with liposomes only and similarly bleeding for anti-bFGF titer.

C. Analysis of Vaccine Potency

a. Antisera from vaccinated mice are analyzed for anti-bFGF activity by ELISA assay.

b. Two tumor models are used for the analysis of anti-tumor therapy: hypercarcinogenic melanoma inducing macroscopic lung tumors 14 days after intravenous inoculation of 10 ⁶ cells in B16F10, C57BL / 6J mice; And Lewis lung carcinoma-low metastatic (LLC-LM) developed by M. O'Reill at Children's Hospital. In addition, the efficacy of the vaccination protocol as an anticancer treatment was evaluated in both allogeneic and genetic syngeneic; The former is a high immunogenic tumor and the latter is non-immunogenic.

Groups of mice vaccinated with vaccinated, non-vaccinated and control liposomes (without bFGF) are challenged intravenously with B16F10 and lung tumors are analyzed on day 14. A large number of tumors visible in 4x magnification microscopy were measured in each group.

The efficacy of the anti-bFGF vaccine is analyzed for both primary tumor growth and subsequent progression of metastatic disease using the LLC-LM model. After 14 days of the last boost, mice are challenged with 10 ⁶ LLC-LM (taken directly from mice with tumors). Tumor volume is measured 14-21 days after inoculation and compared between groups. At this time (14-21 days after tumor inoculation) the tumor is excised from half of the group and the other half is subjected to surgery. Mice were beheaded 14 days after surgery, lungs were excised and weighed and lung metastases counted using a stereomicroscope.

result

Preliminary data from bFGF-liposomal-vaccinated and control liposome-vaccinated Balb / cByJ mice indicate that bFGF vaccine stimulates anti-bFGF antibody response. Anti-bFGF titers range from 1: 12,800 to 1: 30,000 for Balb / cByJ mice vaccinated in two separate experiments (FIGS. 10A and 10B). Likewise, C57BL / 6J mice respond to vaccination with anti-bFGF titers of 1: 10,000 to 1: 50,000 measured in each mouse (FIG. 13).

Immune Balb / cByJ mice are challenged with LLC-LM (allogeneic) and primary tumor development is evaluated over a 21 day period. The mean tumor volume of control liposome-vaccinated mice is 830 mm ³ compared to 290 mm ³ of similarly challenged bFGF-liposome-vaccinated mice 14 days after tumor implantation (FIG. 14). T / C = 0.35 in this experiment (T / C refers to the number of pulmonary nodules in the vaccinated group divided by the number of nodules in the control vaccine (liposomes only, no peptide)); Tumor size was reduced by 65% compared to the control.

In a second experiment with LLC-LM of vaccinated and non-vaccinated Balb / cByJ mice, the mean tumor volume of control liposome-vaccinated mice was 111 of bFGF-liposome-vaccinated mice similarly challenged 14 days after tumor transplantation. 229 mm ³ as compared to mm ³ (FIG. 15). T / C = 0.48 for this experiment; Tumor size was reduced by 52%.

Balb / cByJ mice are allogeneic to LLC-LM and tumor size in all groups is smaller than that observed in genetically homologous C57BL / 6J mice. Thus, the next set of experiments included (a) LLC-LM (primary tumor development and metastatic disease assessment); Or (b) vaccinated C57BL / 6J mice challenged with B16F10 (measured metastasis); All are genetic kinetic models.

As shown in FIG. 13, C57BL / 6J mice respond to vaccination with the production of bFGF antibodies. To assess the protective efficacy of bFGF antibodies induced in hereditary syngeneic tumors, vaccinated mice are challenged with LLC-LM and primary tumor growth and subsequent metastatic disease are evaluated. The resulting circulating bFGF antibody in response to vaccination and bFGF at this genetic linkage does not alter the growth of primary LLC-LM in C57BL / 6J mice.

Primary tumors of vaccinated mice are surgically resected to assess the extent of metastatic disease following removal of the primary tumor. In the metastatic disease of the LLC-LM model, removal of the primary tumor results in the rapid growth of metastatic tumors of the lung at the same time as the lung weight increases. Vaccination does not affect the development of metastases after removal of the primary tumor, as confirmed by lung weight measured 14 days after primary tumor removal.

Finally, C57BL / 6J mice are vaccinated and challenged intravenously with B16F10 to assess vaccine efficacy in a genetic syngeneic model of experimental metastasis. The anti-bFGF titer of vaccinated mice is 1: 16,000, but B16F10 metastasis in the lungs of bFGF-liposome vaccinated mice is not different from the metastasis in the lungs of mice vaccinated with control liposome preparations or control mice (PBS inoculation). not.

conclusion

The data presented in this experiment demonstrate that vaccine compositions comprising growth factors, particularly fibroblast growth factors, can be effectively used to prevent or eliminate cancerous tumors, especially allogeneic cancerous tumors or highly immunogenic tumors. As shown in both parts of the experiment, mice immunized with fibroblast growth factor introduced into liposomes and challenged with allogeneic tumors show an average 65% and 52% reduction in tumor size compared to the control immunized with the control vaccine. . In addition, the antibody titers of mice receiving a vaccine composition containing growth factors are significantly greater than mice not receiving a vaccine.

Example 12

Efficacy of bFGF Heparin Binding Domain Peptides on the Growth of B16BL6 or HUVEC Tumor Cells in Vitro

As described in Example 9, the heparin binding domain peptide of bFGF shows inhibitory effect on bFGF stimulated HUVEC proliferation in vivo. The same efficacy was not seen when incubated with tumor cells in vitro.

Cell line

Human umbilical vein endothelial cells (HUVEC) were cloned into 75 cm 2 cell-culture flasks, 37 ° C., humid air, 10% CO ₂ to 1% L-glutamine (BioWhittaker, Walkersville, MD) and cerebellar extracts. Maintain in supplemented endothelial growth medium (EGM-2, Clonetics, Walkersville, MD).

B16BL6 modified cell lines of B16-F10 melanoma are obtained from the National Cancer Institute-Central Repository (Frederick, MD). Tumor cells in DMEM (BioWhittaker, MD) supplemented with 5% heat inactivated fetal bovine serum and L-glutamine (BioWhittaker, Walkersville, MD) as monolayer cultures in a humid atmosphere of 37 ° C., 5% CO ₂ /95% air. Raise Cells are passaged at 80% aggregation and passages 6-18 are used. B16BL6 has been demonstrated to be free of mycoplasma and the following pathogenic murine viruses: tertiary leo virus, pneumonia virus of mouse, K virus, taylor encephalitis virus, Sendai virus, minute virus of mouse, mouse adenovirus, mouse Hepatitis virus, lymphocytic choroidal meningitis virus, actromelia virus and lactate dehydrogenase virus.

Lewis lung carcinoma-low metastatic (LLC-LM) cell line The same mycoplasma provided by Judah Folkman (Children's Hospital, Boston) and the aforementioned pathogenic rat virus were demonstrated to be absent. LLC-LM is maintained in DMEM supplemented with 10% heat inactivated fetal bovine serum and 1% L-glutamine.

Proliferation assay

HUVECs are washed with PBS at passages 2-5 and trypsinized with 0.05% solution of trypsin-versen mixture (BioWhittaker, Walkersville, MD). Endothelial cell basal medium (EBM-2, Clonetics, Walkersville, supplemented with 2% heat inactivated fetal bovine serum (Hyclone, Logan, UT) and 2 mM L-glutamine at a concentration of 2.5 × 10 ⁴ cells / ml And suspended in 96 well (100 μl / well) culture plates (Costar, Cambridge, Mass.) And incubated at 37 ° C., 5% CO ₂ for 24 hours. Cells were incubated with 1, 10 and 100 μg / ml FGF-2 peptide (22 amino acid control peptides) for 20 minutes or in medium alone, followed by human recombinant FGF-2 (R & D Systems, Minneapolis, MN) for 72 hours. Stimulation at 10 ng / ml or with media alone. Cell proliferation is measured by BrdU introduction using ELISA assay (Boehringer Mannheim, Indianapolis, TN) according to the manufacturer's instructions.

Proliferation assays with B16BL6 or LLC-LM are performed in the following manner. Cells are suspended in Dulbecco's minimal essential medium (DMEM, BioWhittaker, MD) supplemented with 5% fetal bovine serum, 1% L-glutamine at a concentration of 2.5 × 10 ⁴ cells / ml, and 24 wells (500 μl / ml) Plate on culture plates (Costar, Cambridge, MA) and incubate at 37 ° C., 5% CO ₂ for 4 hours. Add FGF-2 peptide at 1, 10, 100 μg / ml and incubate for 20 minutes. After incubation, FGF-2 (10 ng / ml) is added to the wells for 48 hours. Cell proliferation is measured by cell count using a Coulter counter (Coulter, Columbia, MD).

In an alternative method, B16BL6 melanoma cells are plated at 10,000 cells / well in 24 well plates and incubated overnight. Heparin binding peptides are added to the wells at various concentrations, followed by the medium containing 5 ng / ml bFGF. BFGF added in the absence of peptide does not increase the proliferation of B16BL6 beyond the media control. In addition, no significant efficacy on the growth of B16BL6 cells was observed when incubated with heparin binding peptides as measured by cell counts. The results are shown in FIG. This example provides a method useful for screening peptides useful in the methods and compositions of the present invention.

Inhibition of endothelial cell proliferation

To further assess the inhibitory activity of the 44 amino acid peptide (SEQ ID NO: 1) corresponding to the heparin binding domain (HBD) of FGF-2 and to analyze the receptor binding domain (RBD), which is an additional functional domain of FGF-2, These peptides at concentrations of 1, 10 or 100 μg / ml are co-cultured with FGF-2, 5 ng / ml in the HUVEC proliferation assay. The addition of FGF-2 alone stimulates the proliferation of HUVECs compared to cells cultured without growth factors. Both HBD and RBD peptides inhibit FGF-2-induced proliferation of HUVECs in a dose dependent manner, with inhibitory concentrations (IC ₅₀ ) of 70 and 20 μg / ml, respectively (FIG. 19). The 22 amino acid control peptides corresponding to the defined regions of human angiostatin did not produce inhibitory effects on FGF-2 induced proliferation of HUVECs.

Example 13

Inhibition of growth and development of LLC-LM metastasis in mice vaccinated with heparin binding domain peptide of bFGF

The efficacy of vaccinating mice with heparin binding domain peptide bFGF in liposome format is measured using the LLC-LM experimental transfer model.

Mice are vaccinated with heparin binding domain peptides of bFGF in liposome format using the same vaccination method as the B16BL6 experiment above (three inoculations three weeks apart). Two weeks after the last boost, mice are challenged intravenously with LLC-LM. On day 17, control (untreated) mice begin to die due to lung tumors. Behead all groups. The lungs are removed and analyzed based on lung weight. Normal lung weight is subtracted from treated and non-treated groups to determine T / C. Vaccination with heparin binding domain peptide inhibits the growth and development of LLC-LM experimental metastasis by 94% compared to the control. These lungs will be photographed and analyzed histologically. As demonstrated by the results of FIG. 11, the growth and development of metastases is significantly inhibited in mice vaccinated with heparin binding domain peptides.

Example 14

Immunogenicity of Vaccine Compositions Containing Growth Factors

Determination of Antibody Titer and Specificity

Pooled serum is obtained from mice treated with L (HBD), L (RBD), L (LA) or PBS. Anti-FGF-2 specific antibodies are measured by enzyme linked immunosorbent assay (ELISA). Briefly, 96 well emulon 4 plates (Dynotech, Chantilly, VA) were prepared using recombinant FGF-2, heparin binding domain peptide of FGF-2, receptor binding domain peptide of FGF-2, vascular endothelial growth factor. (VEGF) (50 μl; 2 μg / ml in carbonate buffer), incubate overnight at 4 ° C. and then block with 3% milk and 0.05% Tween 20 (PBS / Tween 20) in PBS. Plates are washed three times with PBS / Tween 20 and incubated for 1 hour with serial dilution of serum in PBS / Tween 20. 1 plate with biotin conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgG and IgM to wash plates and measure biotin-conjugated goat anti-mouse immunoglobulin (Ig) G and IgM (K & P) or subclass response. Incubate at RT for hours. After washing, the wells are incubated with hose-radish peroxidase (HRP) -conjugated streptavidin. Optical density is read at 540 nm. Antibody titers are determined by the inverse of the serum dilution giving an O.D. value that is at least 2 standard deviations above the mean O.D. of normal mouse serum.

Example 15

In vivo studies to explain immunogenic efficacy of VEGF peptides in mice

This example involves the use of a vaccine composition comprising growth factor peptides, in particular vascular endothelial growth factor (VEGF) peptide fragments, introduced into liposomes to be immunogens against growth factor peptides when the composition is administered to humans or animals. . Successful immunogen response is achieved in mice when VEGF peptides are introduced into liposomes via active peptide conjugation or passive encapsulation.

A. VEGF Peptides

Three different murine VEGF peptides, named peptide C-E, are introduced into liposomes. Peptide C (SEQ ID NO: 3) is a carboxyl terminated peptide consisting of residues 110-115, exon of 6 amino acids and amino-terminal cysteine for crosslinking. Peptide D (SEQ ID NO: 4) is also a carboxyl terminated peptide containing residues 102-115, exon of 6 amino acids and amino-terminal cysteine for crosslinking. Peptide E (SEQ ID NO: 5) is an amino terminal peptide corresponding to 1-20 residues of VEGF with C-terminal cysteine added for crosslinking. The amino acid sequence of each peptide is shown below:

Peptide C CRTKPEKCDKPRR (SEQ ID NO: 3)

Peptide D CECRPKKDRTKPEKCDKPRR (SEQ ID NO: 4)

Peptide E APTTEGEQKSHEVIKFMDVYC (SEQ ID NO: 5)

B. Vaccine Preparation Using Liposomes

Iii. Active peptide conjugation

One method used to introduce growth factor peptides into liposomes involves chemical coupling of peptides to amino acids via thiol groups. In this study, thiol-reactive liposomes are produced by using the homolog of cholesterol, PDS-CHOL. PDS-CHOL is described by White et al. It was first produced according to the method of Vaccine 12 (2): 1111-22 (1995). PDS (2 ', 2'-dipyridylsulfide or Aldrithiol-2 from Aldrich Chem) in a separate vial, while dissolving thiocholesterol (Aldrich Chem., # 13,611-5 Milwaukee, Wisconsin) (1.772 g) in chloroform ) (1.982 g) is dissolved in a 100: 1 mixture of chloroform: glacial acetic acid. Thiocholesterol is added dropwise to PDS which actively mixes at a rate of approximately 30 drops per minute. The vial is then purged with nitrogen, sealed, covered with foil and mixed overnight at room temperature. After a stream of nitrogen was used to evaporate the solvent, the sample was recrystallized by addition of 50 ml of ethanol at approximately 56 ° C., the sample was placed on ice for 1 hour and then at −20 ° C. for 2 days. The sample is recovered by drying with ice-cold ethanol and dried. Purity is measured at 90-95% using thin layer chromatography and iodine vapor.

Once PDS-CHOL was prepared, the lipids, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol (CHOL) and lipid A and 9: 1: 7.4: 2: And a molar ratio of 0.02 (DMPC: DMPG: Cholesterol: PDS: Chol: Lipid A) (mol: mol). The lipid mixture is rotary evaporated with nitrogen and dried. All manipulations are carried out anaerobicly with degassing liquid except for the final PBS wash. The peptide is then dissolved in 10 mM acetic acid to a concentration of 5 mg / ml and 1 mg of equivalent is diluted with 6.4 ml of deionized water. This peptide hydration solution is added to the dried lipids, resuspended via crude sonication, briefly vortexed and then mixed with an orbital shaker overnight at room temperature. The mixture is then lyophilized. Liposomes are preformed by adding 2.135 mL of citrate / phosphate buffer (0.1 M) and vortexing for 1-2 minutes. Additional peptides are dissolved in 10 mM sterile acetic acid at a concentration of 10 mg / ml and 0.2 ml (2 mg) is added to the preformed vesicles. The mixture is covered with foil and mixed overnight with an orbital shaker at room temperature. After washing twice with PBS, liposomes are analyzed by introduction with BCA (Bicinchroninic acid, Pierce Chemical Co. Rockford, Illinois) (for descriptions of the assays used Bartlett et al. J. Bio. Chem. 234: 466-68 (1959). The results of the introductory assay indicate that 68%, 74% and 66% conjugation of the peptides C, D and E occurred, respectively.

ii. Manual encapsulation

Another method used to introduce peptides of growth factors such as VEGF is manual or simple encapsulation. Repeatedly, the lipids are dispensed into pyrogen-free 100 mL round bottom flasks in the following order: lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol. The lipid is rotary evaporated to remove the bulk solvent and then dried. To hydrate the lipid, 4.5 ml of sterile deionized water is added and the mixture is sonicated and left for 2 hours at room temperature. After transfer to the vaccine vial, the mixture is lyophilized. Peptide solution (2.5 mg / ml in room temperature PBS from 10 mg / ml stock in acetic acid) is added to the lyophilized lipid and the vial is vigorously vortexed for 2 minutes. The final concentration for phospholipids is approximately 100 mM. To allow encapsulation, the vial is left at 4 ° C. for 24 hours and then washed twice with PBS. Subsequent analysis of encapsulation indicates that 33%, 19% and 19% encapsulation of the peptides C, D, and E occurred, respectively.

C. Analysis of Vaccine Potency

Five Balb / cByJ mice, 6-8 weeks of age, are immunized by intraperitoneal injection of liposomes into which one of the three peptides described above of VEGF has been introduced via active conjugation or passive encapsulation. The target dose is approximately 10-50 μg peptide and 100 μg lipid A. Serum is collected on day 35 and analyzed for anti-VEGF activity by ELISA. As a control, mice not injected with liposomes are also bleeding for anti-VEGF titer analysis.

Preliminary data from VEGF-peptide-liposomal-vaccinated and control Balb / cByJ mice indicate that VEGF vaccine stimulates anti-VEGF antibody response. Once the peptide is conjugated to liposomes, each Balb / cByJ mouse responds with a titer of at least 1: 100,000 anti-VEGF peptide titers for peptides 2 and 3 and a titer of 1: 25,000 for peptide C (FIG. 16 (a)). -(c)). For peptides introduced by simple encapsulation, each Balb / cByJ mouse responds to peptides D and E with anti-VEGF peptide titers ranging from 1: 12,500 to 1: 50,000 (Figs. 17 (a)-(c)). ).

The above experiments reasonably demonstrate that vaccine compositions comprising peptide epitopes of growth factors, particularly vascular endothelial growth factors, introduced into liposomes by encapsulation or conjugation, can elicit significant antibody responses in vivo.

Example 16

Inhibition of growth and development of B16BL6 experimental metastasis in mice vaccinated with VEGF peptide in liposome format

The synthetic peptide (peptide F, SEQ ID NO: 6) generated with the neurophylline receptor binding domain of VEGF is covalently conjugated to liposome vesicles containing lipid A. Mice are vaccinated with peptide F liposome preparations using the same vaccine method as the bFGF peptide vaccine. Two weeks after the last boost, mice are challenged intravenously with B16BL6. Mice are beheaded 14 days after challenge. Vaccination with peptide F liposome formulation results in significant inhibition of B16BL6 experimental transfer compared to liposome lipid A control (T / C = 0.33). Heparin binding domain peptide bFGF covalently bound to liposomes is used as a positive control in this experiment. The results of this experiment are shown in FIGS. 11 and 18.

Although the invention has been described in more detail with reference to the described embodiments, it will be understood that various modifications and changes may be made within the spirit and scope of the invention.

Claims (46)

An immunogenic composition comprising an immunogenic peptide fragment of fibroblast growth factor and a pharmaceutically acceptable carrier. The composition of claim 1, wherein the immunogen peptide corresponds to the heparin binding domain of the fibroblast growth factor. The composition of claim 1, wherein the amino acid sequence of the immunogen peptide comprises SEQ ID NOs: 1 and 2. The composition of claim 1, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. The method of claim 4, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, tyroglobulin, serum albumin, gamma globulin, cells of genetic syngeneicity and D- and / or A composition comprising a polymer of L-amino acids. The composition of claim 4 further comprising an adjuvant, preservative, diluent, emulsifier and stabilizer. 7. The method of claim 6 wherein the adjuvant is a lipophilic muramyl dipeptide derivative, a nonionic block polymer, aluminum hydroxide, aluminum phosphate, lipid A, incomplete Freund's adjuvant, complete Freund's adjuvant, polydisperse β- (1,4 Bound acetylated met, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin induction adjuvant, killed Bordetella pertussis , polysaccharides of gram-negative bacteria, polymer anion, dextran sulfate, inorganic gel, alum, aluminum hydroxy The composition selected from the group consisting of side and aluminum phosphate. The composition of claim 1 further comprising a hydrophobic moiety attached to an immunogen peptide. 9. The composition of claim 8, wherein the hydrophobic moiety comprises at least one long chain fatty acid having at least 10 carbon atoms in the lipid backbone. 9. The composition of claim 8, wherein the hydrophobic moiety is selected from the group consisting of palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid and linolenic acid. An immunogenic composition comprising an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier. The composition of claim 11, wherein the immunogen peptide fragment corresponds to the receptor binding domain of vascular endothelial growth factor. The composition of claim 11, wherein the amino acid sequence of the immunogen peptide comprises SEQ ID NOs: 3-9. The composition of claim 11, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. 15. The method of claim 14, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, tyroglobulin, serum albumin, gamma globulin, cells of genetic syngeneicity and D- and / or A composition comprising a polymer of L-amino acids. The composition of claim 14 further comprising an adjuvant, preservative, diluent, emulsifier and stabilizer. The method of claim 16, wherein the adjuvant is a lipophilic muramyl dipeptide derivative, a nonionic block polymer, aluminum hydroxide, aluminum phosphate, lipid A, incomplete Freund's adjuvant, complete Freund's adjuvant, polydisperse β- (1,4 Bound acetylated met, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin induction adjuvant, killed Bordetella pertussis , polysaccharides of gram-negative bacteria, polymer anion, dextran sulfate, inorganic gel, alum, aluminum hydroxy The composition selected from the group consisting of side and aluminum phosphate. 15. The composition of claim 14, further comprising a hydrophobic moiety attached to an immunogen peptide. 19. The composition of claim 18, wherein the hydrophobic moiety comprises at least one long chain fatty acid having at least 10 carbon atoms in the lipid backbone. 19. The composition of claim 18, wherein the hydrophobic moiety is selected from the group consisting of palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid and linolenic acid. An immunogenic composition comprising a fibroblast growth factor and an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier. The composition of claim 21, wherein the immunogenic peptide fragment of the fibroblast growth factor corresponds to the heparin binding domain of the fibroblast growth factor and the immunogenic peptide fragment of the vascular endothelial growth factor corresponds to the receptor binding domain of the vascular endothelial growth factor. . The composition of claim 22, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. The method of claim 23, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, tyroglobulin, serum albumin, gamma globulin, cells of genetic syngeneicity and D- and / or A composition comprising a polymer of L-amino acids. The composition of claim 23 further comprising an adjuvant, preservative, diluent, emulsifier and stabilizer. The adjuvant of claim 25, wherein the adjuvant is a lipophilic muramyl dipeptide derivative, a nonionic block polymer, aluminum hydroxide, aluminum phosphate, lipid A, incomplete Freund's adjuvant, complete Freund's adjuvant, polydisperse β- (1,4 Bound acetylated met, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin induction adjuvant, killed Bordetella pertussis , polysaccharides of gram-negative bacteria, polymer anion, dextransulfate, inorganic gel, alum, aluminum hydroxy The composition selected from the group consisting of side and aluminum phosphate. A method of treating cancer or hyperproliferative disorders in humans or animals comprising administering to a human or animal an effective amount of a composition comprising an immunogenic peptide fragment of a fibroblast growth factor and a pharmaceutically acceptable carrier. The method of claim 27, wherein the immunogen peptide fragment corresponds to the heparin binding domain of the fibroblast growth factor. The method of claim 27, wherein the amino acid sequence of the immunogen peptide fragment comprises SEQ ID NOs: 1 and 2. The method of claim 27, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. 28. The method of claim 27, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, tyroglobulin, serum albumin, gamma globulin, cells of genetic syngeneicity and D- and / or A method comprising a polymer of L-amino acids. The method of claim 27 further comprising an adjuvant, preservative, diluent, emulsifier and stabilizer. 28. The method of claim 27, wherein the hyperproliferative disorder is angioma, solid tumor, blood mediated tumor, leukemia, metastasis, capillary dilatation, psoriasis, scleroderma, purulent granuloma, myocardial angiogenesis, Crohn's disease, plaque angiogenesis, arteriovenous malformation, Corneal disease, redness, neovascular glaucoma, diabetic retinopathy, posterior capsular fibrosis, arthritis, diabetic angiogenesis, macular degeneration, wound union, peptic ulcer, Helicobacter related disease, fracture, keloid, angiogenesis, hematopoiesis, ovulation, Methods including menstruation, placental formation and cerebellar fever. A method of treating cancer or hyperproliferative disorders in humans or animals comprising administering to a human or animal an effective amount of a composition comprising an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier. The method of claim 34, wherein the immunogen peptide fragment corresponds to the receptor binding domain of vascular endothelial growth factor. The method of claim 34, wherein the amino acid sequence of the immunogen peptide fragment comprises SEQ ID NOs: 3-9. 35. The method of claim 34, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. The method of claim 34, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, tyroglobulin, serum albumin, gamma globulin, cells of genetic syngeneicity and D- and / or A method comprising a polymer of L-amino acids. 35. The method of claim 34, further comprising an adjuvant, preservative, diluent, emulsifier, and stabilizer. 35. The method of claim 34, wherein the hyperproliferative disorder is hemangioma, solid tumor, blood mediated tumor, leukemia, metastasis, capillary dilatation, psoriasis, scleroderma, purulent granuloma, myocardial angiogenesis, Crohn's disease, plaque angiogenesis, arteriovenous malformation, Corneal disease, redness, neovascular glaucoma, diabetic retinopathy, posterior capsular fibrosis, arthritis, diabetic angiogenesis, macular degeneration, wound union, peptic ulcer, Helicobacter related disease, fracture, keloid, angiogenesis, hematopoiesis, ovulation, Methods including menstruation, placental formation and cerebellar fever. Administering to a human or animal an effective amount of a growth factor composition that renders the composition immunogenic to the fibroblast growth factor when the composition comprises an immunogenic peptide fragment of the fibroblast growth factor and a pharmaceutically acceptable carrier A method of treating a human or animal in need of an immune response to a growth factor comprising the step of. 42. The method of claim 41, wherein the immunogen peptide comprises SEQ ID NOs: 1 and 2. 42. The method of claim 41, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins. An effective amount of growth factor composition is administered to a human or animal, such that the composition comprises an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier to render it immunogenic to the vascular endothelial growth factor when administered to a human or animal. A method of treating a human or animal in need of an immune response to a growth factor comprising the step of. 45. The method of claim 44, wherein the immunogen peptide comprises SEQ ID NOs: 3-9. 45. The method of claim 44, wherein the pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier proteins.