JP2002538219A - Compositions and methods for treating cancer and hyperproliferative diseases - Google Patents

Abstract

  (57) [Summary] Compositions and methods are provided that are effective in eliciting an immune response to inhibit abnormal or unwanted cell proliferation, particularly endothelial cell proliferation and angiogenesis associated with angiogenesis and tumor growth. The compositions of the present invention comprise a naturally occurring or synthetic protein, peptide, or protein fragment containing all or an active portion of a growth factor in a pharmaceutically acceptable carrier. Preferred growth factors include basic fibroblast growth factor and vascular endothelial growth factor. The method of the invention involves administering to a human or animal a composition described herein at a dosage sufficient to elicit an immune response. The methods of the present invention are useful for treating unwanted and uncontrolled cell growth, eg, diseases and processes caused by cancer, particularly where uncontrolled cell growth is affected by the presence of growth factors. Administration of a composition of the present invention to a human or animal having a metastatic tumor is useful for preventing the growth or spread of such tumors.

Description

DETAILED DESCRIPTION OF THE INVENTION

CROSS REFERENCE TO RELATED APPLICATIONS The present invention relates to US Provisional Patent Application No. 60/07, filed March 10, 1998.
U.S. Patent Application No. filed on March 11, 1999, which is not a provisional application for 7,460, but is a continuation-in-part of U.S. Patent Application No. 09 / 264,213 filed on March 10, 1999 09 / 266,453, which is a continuation-in-part application and its priority application.

TECHNICAL FIELD The present invention relates to methods and compositions for preventing or reducing human or animal cancer. More particularly, the present invention relates to immunogenic compositions comprising growth factors, active fragments thereof, antibodies specific for the growth factors and methods of using them.

BACKGROUND OF THE INVENTION [0003] While many cancers can be treated with chemotherapeutic agents, a significant number of cancers are inherently drug resistant, and others develop resistance during or after chemotherapy. I do. Cancer is often resistant to one or more types of drugs. This phenomenon is called multidrug resistance or MDR. Accordingly, there is a great need for compositions and methods that can be used in addition to or as an alternative to chemotherapy for treating cancer.

[0004] A major clinical problem in cancer is metastasis. By the time the primary tumor is identified and localized,
Seed cells often leak and move or metastasize to other organs in the body,
And there a secondary tumor is established. Many 2 even after removing the primary tumor
Surgical methods are rarely sufficient for the cure of cancer, as the secondary tumor survives and grows. As a result, there is an immediate and urgent need for techniques to eradicate already existing secondary tumors.

[0005] Cancer cells that escape from the primary tumor are usually carried by the veins and lymphocyte circulation, where they subsequently settle in the downstream capillary beds or lymph nodes. However, if only one of the 10,000 cancer cells leaked from the primary tumor is present, a secondary tumor will be established. Cancer cells that successfully establish a secondary tumor are cells that have found a favorable environment for survival and growth. This preferred environment includes hormones and growth promoting factors. Stimulators include local growth factors, hormones produced by the host, and self-stimulating growth factors produced by the tumor cells themselves. Thus, there is an immediate and urgent need for technologies that can prevent or inhibit the spread of cancer and the formation of secondary tumors.

[0006] In addition, there are a number of other hyperproliferative diseases. Hyperproliferative diseases are caused by non-cancerous (ie, non-neoplastic) cells that overproduce in response to certain growth factors. Examples of such hyperproliferative disorders include diabetic retinopathy, psoriasis, endometriosis, macular degenerative disorders and benign proliferative disorders such as prostatic hypertrophy and lipomas.

[0007] Many new cancers are initiated by growth factors (ie, angiogenic factors) that affect either the cancer cells themselves or normal cells around the cancer and help the cancer cells survive, and existing cancers And hyperproliferative diseases are known to be stimulated. There is a direct correlation between circulating levels of certain growth factors and cancer growth. A potential treatment modality would be to regulate the levels of circulating growth factors in the patient to prevent the growth or recurrence of the cancer, and to reduce or eliminate existing cancer. Therefore, what is needed is a composition that removes a target growth factor from the circulation or inhibits the growth promoting activity of the growth factor.

[0008] Cell proliferation is a normal ongoing process in all living organisms, and involves a number of factors and signals that are finely balanced to maintain a regular cell cycle. is there. The general process of cell division consists of two sequential processes: nuclear division (mitosis) and cytokinesis (cytokinesis). As organisms are continuously growing and replacing cells, cell proliferation is a central process essential for the normal functioning of almost all biological processes. Whether mammalian cells proliferate and divide is determined by various feedback control mechanisms,
And these mechanisms include the availability of gaps where cells can grow and the secretion of specific stimuli and inhibitors in the immediate environment.

[0009] If normal cell growth is hindered or somehow disrupted, a series of biological functions can be affected as a result. Growth disruption may be due to a myriad of factors, such as the lack or excess of various signal-forming drugs, growth factors or the presence of environmental changes. Diseases characterized by abnormal cell proliferation include cancer, abnormal embryonic development, inappropriate luteal formation, difficulty in wound healing, and dysfunction of the inflammatory and immune responses.

[0010] Cancer is characterized by abnormal cell growth. Cancer cells exhibit a number of properties that can be dangerous to the host, and these properties often include the ability to invade other tissues and induce capillary ingrowth, and thereby promote the growth of Cancer cells ensure adequate blood supply. One of the distinctive features of cancer cells is that cancer cells respond abnormally to regulatory mechanisms that regulate normal cell division and continue to divide relatively uncontrolled until they kill the host That's what it means.

[0011] Angiogenesis and angiogenesis-related diseases are closely influenced by cell proliferation and, therefore, cytokines and growth factors. As used herein, the term "angiogenesis"
Means that new blood vessels form in the tissue or organ. Under normal physiological conditions, humans or animals undergo angiogenesis only under very specific restricted circumstances. For example, angiogenesis is usually observed in wound healing, fetal and embryonic development, and formation of the corpus luteum, endometrium and placenta. The term "endothelium" is defined herein as a thin layer of squamous cells lining the serosal cavity, lymphatic vessels and blood vessels. These cells are defined herein as "endothelial cells." The term "endothelial inhibitory activity" refers to the possibility that a molecule will generally inhibit angiogenesis. Inhibition of endothelial cell proliferation also results in inhibition of angiogenesis.

It is believed that both controlled and uncontrolled angiogenesis proceed in a similar manner. Endothelial cells and pericytes surrounded by the basement membrane form capillaries. Angiogenesis begins with erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells lining the lumen of the blood vessel then protrude from the basement membrane. Angiogenic stimulators induce endothelial cells to migrate from the eroded basement membrane. The migrating cells form a "sprout" apart from the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. Endothelial sprouts combine with each other to form capillary loops, creating new blood vessels.

[0013] Persistent uncontrolled angiogenesis occurs in a number of disease states, tumor metastasis and abnormal proliferation by endothelial cells, and supports the pathological damage seen in these conditions. The various pathological disease states in which uncontrolled angiogenesis exists are classified together as angiogenesis-dependent, angiogenesis-related, or angiogenesis-related diseases. These diseases are the result of abnormal or unwanted cell proliferation, especially endothelial cell proliferation.

The hypothesis that tumor growth is angiogenesis-dependent is based on the conclusion of Judah Folkman (N. Engl. Jo
ur. Med. 285: 1182 1186, 1971), first proposed in 1971. In its simplest language, this hypothesis proposes that expansion of tumor volume beyond a certain phase requires induction of new capillaries. For example, pulmonary micrometastasis of the early prevascular phase in mice would not be detectable except by high-performance microscopy on histological sections. Further indirect evidence supporting the notion that tumor growth is angiogenesis-dependent is provided in U.S. Patent Nos. 5,639,725, 5,629,327, 5,792,845; 5,733,876 and 5,854,205, all of which are incorporated herein by reference.
Seen inside.

[0015] Recent research in the field of angiogenesis has led to the current paradigm suggesting that phenotypic changes associated with tumor-associated angiogenesis are effected by an angiogenic switch. In the homeostatic state, an equilibrium between inhibitors of angiogenesis and stimulators is maintained. In contrast, during overgrowth, this balance is shifted towards the angiogenic phenotype. This switch may be used to upregulate endogenous angiogenesis stimulators, such as fibroblast growth factor, vascular endothelial growth factor or interleukin, or downregulate angiogenesis inhibitors, such as angiostatin or endostatin protein. May be brought by either.

One example of a disease resulting from angiogenesis is an angiogenic disease of the eye.
The disease is characterized by the invasion of new blood vessels into eye structures such as the retina or cornea. It is the most common cause of blindness and is involved in roughly 20 eye diseases. In age-related macular degeneration, a related visual problem is caused by the ingrowth of choroid capillaries from defects in the Brook's membrane and the proliferation of fibrovascular tissue beneath the retinal pigment. Angiogenic damage has also been associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, and retro lens fibroplasia. Other diseases associated with corneal neovascularization include epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior marginal keratitis, dry pterygitis, sjogrens, erythematous Acne, phylectenulosis, syphilis, mycobacterial infection, lipid degeneration, chemical burn, bacterial ulcer, fungal ulcer, herpes simplex infection, herpes zoster infection, protozoan infection, Kaposi's sarcoma, Mohren Ulcers, Terrien's limbal degeneration, mariginal epidermolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's sarcomatosis, sclerosis, Stevens Johnson's disease and limbic Includes, but is not limited to, radial keratotomy.

Diseases associated with retinal / choroidal neovascularization include diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, elastic fibrous pseudoxanthoma, Paget's disease, venous obstruction, Infections that cause arterial occlusion, carotid artery occlusion, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, prematurity retinopathy, Eils disease, Behcet's disease, retinitis or choroiditis Disease, presumed ocular histoplasmosis, best disease, myopia, orbit, Stargardt's disease, planaritis, chronic retinal detachment,
These include, but are not limited to, hyperviscosity syndrome, toxoplasmosis, trauma and post-laser complications. Other diseases include those associated with rubeosis (angiogenesis of the horn) and those caused by the overgrowth of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy. Not limited.

Another disease that may be involved in angiogenesis is rheumatoid arthritis. Angiogenesis forms blood vessels in the synovium lining the joint. In addition to forming new vascular networks, endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. These factors involved in angiogenesis appear to actively contribute to and help maintain the chronic inflammatory state of rheumatoid arthritis.

Factors associated with angiogenesis may also have a role in osteoarthritis. Activation of chondrocytes by angiogenesis-related factors contributes to joint destruction. At a later stage, leg tube forming factors will promote new bone formation. Treatment to prevent bone destruction could halt disease progression and reduce the symptoms of arthritis patients.

[0020] Chronic inflammation may also be involved in pathological angiogenesis. Disease states such as ulcerative colitis and Crohn's disease show histological changes, and new blood vessels grow inward into the inflamed tissue. Bartonellosis, a bacterial infection found in South America, can produce a chronic stage characterized by proliferation of vascular endothelial cells. Another pathological role associated with angiogenesis is found in atherosclerosis. Plaques formed in the lumen of blood vessels have been shown to have angiogenic stimulatory activity.

[0021] One of the most frequent angiogenic diseases of children is hemangiomas. In most cases,
The tumor is benign and regresses without intervention. In more severe cases, the tumor progresses to large spongy and infiltrating forms and produces clinical complications. The general form of hemangiomas, ie, hemangiomatosis, has a high mortality rate. There are refractory hemangiomas that cannot be treated with currently used therapies.

Angiogenesis is also responsible for the disorders found in genetic diseases such as Osler-Weber-Rangeux disease or hereditary hemorrhagic peripheral vasodilation. It is a genetic disease characterized by a number of small hemangiomas, vascular or lymphatic tumors. Hemangiomas are found on the skin and mucous membranes, often with epistaxis (nasal bleeding) or gastrointestinal bleeding, and sometimes with pulmonary or hepatic arteriovenous fistulas.

Thus, it is clear that cell proliferation, especially endothelial cell proliferation, plays a major role in cancer pathology and in the growth and development of metastases. If it is possible to suppress, inhibit or eliminate this abnormal or undesired proliferative activity, the tumor will be present but will not grow. In disease states, abnormal or undesirable cell proliferation and angiogenesis could be prevented, thereby avoiding the disorders caused by the invasion of new microvasculature. Therapies aimed at controlling the cell proliferation process could lead to the termination or mitigation of these diseases.

Therefore, what is needed are compositions and methods that can inhibit abnormal or undesirable cell growth associated with a tumor. These compositions must be able to overcome the activity of endogenous growth factors in pre-metastatic tumors and prevent the spread of cancer cells, thereby inhibiting disease development and tumor growth. These compositions must also be able to regulate the formation of capillaries during the angiogenesis process. Finally,
Compositions and methods for inhibiting cell proliferation are preferably non-toxic and should produce few side effects.

SUMMARY OF THE INVENTION The present invention generally includes methods and compositions for preventing or treating cancer.
More particularly, the present invention relates to compositions containing immunogenic growth factors, including growth factors or active fragments thereof, optionally in combination with a delivery vehicle.
Without wishing to be bound by the following theory, the compositions of the present invention can elicit either a cellular response or an immune response that results in the prevention and reduction of cancer.

The present invention provides a method of vaccinating a human or animal against a growth factor associated with a particular cancer type and hyperproliferative disease. Some cancers are associated with only one growth factor, while others are regulated by several growth factors. For example, some T-cell lymphomas produce the growth factor IL-2, which stimulates growth by an autocrine effect. Other tumors, on the other hand, produce factors that promote angiogenesis by inducing tissue angiogenesis at the site of metastasis and stimulate the growth of metastatic cancerous lesions.

The growth factor-containing composition of the present invention may be a liposome or vesicle in which a portion of a growth factor, a growth factor fragment, a synthetic peptide of a certain epitope of the growth factor or a modified growth factor fragment is displayed on the outer surface. A suitable delivery or carrier medium can optionally be included. In an alternative embodiment, the growth factor or immunogenic fragment thereof may be partially or completely encapsulated in a carrier such as a liposome. In another alternative embodiment of the invention, the growth factor or active portion thereof comprises:
It can be transported to the desired site by a delivery mechanism involving the use of a colloidal metal such as gold colloid. The composition described above is useful as a vaccine that would otherwise be recognized as "self" by the immune system and induce immunity against growth factors that are not naturally antigenic. The compositions of the present invention are further useful in therapeutic regimes that reduce tumor cell proliferation. Without wishing to be bound by the following theory, the resulting antibodies in the blood circulation bind to growth factors and thereby prevent the onset of cancer growth, reduce existing cancer, or reduce cancer spread. It is considered to inhibit.

[0028] The invention also includes an isolated recombinant antibody specific for a growth factor. Isolated antibodies are produced by and purified from humans or animals with a strong immune system, and are useful for humans or animals with a weak or non-functional immune system in need of such circulating antibodies. Injected. Thus, in accordance with the present invention, cancer is reduced by either actively immunizing an individual using an antigenic growth factor containing composition or passively immunizing by administering an antibody specific for a growth factor epitope. Or be inhibited. In addition, the patient is immunized with the growth factor composition of the invention before the onset of cancer or relapse after cancer treatment.

Accordingly, it is an object of the present invention to provide methods and compositions that reduce cancer and inhibit tumor growth in humans or animals with cancer.

Another object of the present invention is to provide methods and compositions for treating and preventing the appearance or spread of cancer.

A further object of the present invention is a method for reducing cancer and inhibiting tumor growth in humans or animals with cancer by eliciting an active cellular and / or humoral response in the host. And a composition.

Another object of the present invention is to provide methods and compositions for reducing and preventing the appearance of hyperproliferative diseases.

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

Yet another object of the present invention is to provide methods and compositions for passively immunizing a human or animal against a selected growth factor.

Another object of the present invention is to provide a growth factor-containing composition that is antigenic in a human or animal and elicits an immune response against the growth factor.

Another object of the present invention is to provide a vaccine composition, wherein the composition comprises a non-immunogenic growth factor in a human or animal immunized with the composition;
The composition comprises a carrier on which the growth factor is presented so that when the composition is administered to a human or animal, it is immunogenic for the growth factor.

Yet another object of the present invention is to provide a growth factor containing composition, wherein said growth factor is fibroblast (FGF), interleukin, keratinocyte growth factor, colony stimulating factor, epithelium Growth factor (EGF), vascular endothelial growth factor (VEGF)
, Transforming growth factor, Schwann cell-derived growth factor, nerve growth factor (
NGF), platelet-derived growth factor (PDGF), insulin-like growth factors 1 and 2 (
IGF-1 and IGF-2), glial growth factor, tumor necrosis factor, prolactin,
Growth hormone and / or active fragments thereof.

Yet another object of the present invention is to provide growth factor peptide fragments and their use that have been modified with an antigenic moiety to enhance an individual's response to growth factors.

[0039] Yet another object of the present invention is to provide a growth factor containing composition wherein the carrier for the 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 vesicle derived from baculovirus.

Yet another object of the present invention is to provide a composition comprising a growth factor peptide fragment in combination with a pharmaceutically acceptable adjuvant to stimulate an immune response.

Still another object of the present invention is to provide hemangiomas, solid tumors, blood-derived tumors, leukemias, metastases, telangiectasia, psoriasis, scleroderma, purulent granulomas, myocardial angiogenesis, Crohn's disease , Plaque angiogenesis, cerebral arteriovenous malformation, corneal disease, rubeosis, angiogenetic glaucoma, diabetic retinopathy, post lens fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, helicobacter-related Compositions and methods for treating diseases and processes caused by angiogenesis, including, but not limited to, diseases, fractures, keloids, angiogenesis, hematopoiesis, ovulation, menstruation, placenta formation, and feline scratching fever. To provide.

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

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

These and other objects, features and advantages of the present invention will become apparent after reviewing the following detailed description of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention can be more readily understood by reference to the following detailed description of specific embodiments included herein. Although the present invention has been described with respect to certain specific embodiments of the invention, such detailed embodiments are not intended to be considered as limitations on the scope of the invention. All references mentioned herein are U.S. patent application Ser. Nos. 09 / 266,453 and 09/264, U.S. Pat.
213, U.S. Provisional Application No. 60 / 077,460 and U.S. Patent No. 5,919,45.
No. 9 is included as such in their entirety.

The present invention includes methods and compositions for preventing or reducing cancer and hyperproliferative diseases in humans or animals. The immunogenic growth factor-containing composition of the present invention includes a growth factor-containing carrier, such as liposomes and vesicles, immunogenic growth factors and their peptide fragments, immunogenic growth factor peptide fragments in combination with adjuvants, Modified growth factor peptide fragments, modified growth factor peptide fragments in combination with adjuvants, carriers containing growth factor peptide fragments, as well as carriers containing modified growth factor peptide fragments are included, but are not limited to. Still further, the present invention includes antibodies and other cellular responses directed to and specific for growth factors. The invention also includes an RNA sequence encoding the protein, and transfecting an animal or human to introduce the antigenic protein into the system and thereby elicit an immune response to the antigenic growth factor in the body. It also includes the use of the above RNA sequence to carry it.

Definitions As used herein, “a,” “an,” and “the” mean one or more, and are defined to include pluralities unless the context is inappropriate. You.

As used herein, the term “antibody” includes monoclonal antibodies, polyclonal, chimeric, single chain, bispecific, monkey and humanized antibodies, and the products of a Fab immunoglobulin expression library. Fab fragments are included.

The term “antigen” refers to one or a fragment thereof capable of inducing an immune response in an animal. The term includes immunogens and regions that contribute to antigenicity or antigenic determinants.

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

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

As used herein, the term “body fluid” includes saliva, gum secretions, cerebrospinal fluid, gastrointestinal fluid, mucus, urogenital secretions, synovial fluid, blood, serum, plasma, urine, cystic fluid But not limited to, lymph, ascites, pleural extravasation, interstitial fluid, intracellular fluid, ocular fluid, semen, mammary secretions, and vitreous and nasal secretions.

The phrase “consisting essentially of” is used herein to exclude any element that substantially alters the essential properties of the peptide to which this phrase refers. Thus, the statement “consisting essentially of” on a peptide excludes any amino acid substitutions, additions or deletions that substantially alter the biological activity of the peptide.

The term “peptide” refers to an amino acid to which the α-carbon is attached by a peptide bond formed by a condensation reaction between the carboxyl group of the α-carbon of one amino acid and the amino group of the α-carbon of another amino acid ( (Typically L-amino acids). The terminal amino acid at one end of the chain (ie, the amino terminus) has a free amino group, while the terminal amino acid at the other end of the chain (ie, the carboxy terminus) has a free carboxyl group. . Thus, the term “amino terminus” (abbreviated as N-terminus) refers to the free α-amino group of an amino acid at the amino terminus of the peptide, or the α amino acid at any other position within the peptide. Refers to an amino group (an imino group when participating in a peptide bond). Similarly, the term "carboxy terminus" (abbreviated C-terminus) refers to the free carboxyl group of an amino acid at the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other position within the peptide Say

Typically, the amino acids making up the peptide are numbered in order, starting at the amino terminus and increasing toward the carboxy terminus of the peptide. Thus, when one amino acid is said to "follow" another amino acid, that 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 an amino acid mimic incorporated into an oligopeptide by an amide bond or amide bond mimic. As such, the amino acid can be a naturally occurring amino acid, or, unless otherwise limited, a known analog of a naturally occurring amino acid that functions in a manner similar to the naturally occurring amino acid (ie, an amino acid mimetic). Product). Furthermore,
The amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

Furthermore, one of skill in the art will alter one or a small percentage of amino acids (typically less than 5%, more typically less than 1%) within the encoded sequence, as described above. Recognize that individual substitutions, deletions or additions that are added, or deleted, are conservatively modified changes in which certain amino acids have been replaced by chemically similar amino acids as a result of these modifications. Will do. Conservative substitution tables providing functionally similar amino acids are well known in the art. The next six groups each
Contains amino acids that are conservative substitutions for each other: 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 normally associated components as found in the natural state. Say Therefore, the peptides described herein usually have these in situ (in s
itu) Does not contain any environment-related substances. Typically, the isolated antiproliferative peptides described herein will have at least about 80%, usually at least about 90%, and preferably at least about 90%, as measured by band intensity on a silver stained gel. 95% pure.

The purity or homogeneity of a protein can be shown by a number of methods well known in the art, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualization by staining. For some purposes, high resolution is needed and HP
LC or similar means of purification would be used.

When the proteins and peptides of the present invention are relatively short in length (ie, less than 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 sequential addition of the remaining amino acids of the sequence is a preferred method for chemical synthesis of antiproliferative peptides described herein. is there. Solid phase synthesis techniques are known to those skilled in the art.

Alternatively, the antigenic peptides described herein are synthesized using recombinant nucleic acid technology methodology. Generally, this creates a nucleic acid sequence encoding the peptide, places the nucleic acid in an expression cassette under the control of a particular promoter, expresses the peptide in a host, and expresses the expressed peptide or polypeptide. Release and, if necessary, regenerating the peptide. Techniques sufficient to teach such methods to those skilled in the art can be found in the literature.

[0065] Once expressed, the recombinant peptide can be purified according to standard methods, including ammonium sulfate fractionation, affinity columns, column chromatography, gel electrophoresis, and the like. For use as a therapeutic agent, substantially pure compositions of about 50-95% homogeneity are preferred, and 80-95% or more homogeneity are most preferred.

Those skilled in the art will recognize that, after chemical synthesis, biological expression or purification, the immunogenic peptide may have a conformation substantially different from the natural conformation of the constituent peptides. Will. In this case, it is often necessary to denature and deform the immunogenic peptide and then regenerate the peptide to the preferred conformation. Methods for deforming and denaturing proteins and inducing regeneration are well known to those skilled in the art.

As used herein, the term “growth factor” refers to growth factors and polypeptide angiogenic factors, and their modified derivatives and active peptide fragments. The term “growth factor” is: fibroblast growth factor (FGF); interleukins 1-12 (IL-1α, β, IL-2, IL-3, IL-4
, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1
Keratinocyte growth factor; colony stimulating factor such as granule cell colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF or CSF-1) and GM-CSF; epidermal growth factor ( EGF); Vascular endothelial growth factor (VEGF, also known as vascular permeability factor); Transforming growth factor α (TGF-α); Transforming growth factor β1-β5 (TGF-β); Schwann cell-derived proliferation Nerve growth factor (NGF); Platelet-derived 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 growth hormone.

As used herein, the term “immunogenic” refers to eliciting or enhancing antibodies, T-cells and other reactive immune cells directed to endogenous growth factors and A substance that contributes to the immune response.

[0069] An individual may have circulating antibodies directed against an endogenous growth factor without having experienced an immune response to the growth factor. Therefore, as used herein, the term “non-immunogenic” usually elicits no or only low values of circulating antibodies, T-cells or reactive immunity, and usually A natural endogenous growth factor that does not elicit an immune response against itself. When the individual produces sufficient antibodies, T-cells and other reactive immune cells against the administered growth factor composition of the invention to alleviate or alleviate the cancer or hyperproliferative disease to be treated. An immune response is generated.

As used herein, the term “carrier” is capable of incorporating or binding to a growth factor or growth factor fragment, and thereby transforming a growth factor or a portion of a growth factor into a human. Or presented or exposed to the animal's immune system,
And a construct that renders the growth factor-carrier composition immunogenic with respect to endogenous growth factor (s). The term "carrier" further includes delivery methods that allow a growth mechanism or growth factor fragment composition to be delivered to a desired site by a delivery mechanism. One example of such a delivery system uses a colloidal metal, such as colloidal gold (see PCT / US94 / 03177, filed March 18, 1994).

In addition, the term “carrier” further includes keyhole limpet hemocyanin (KLH
), Bovine serum albumin (BSA) and other adjuvants, including but not limited to vaccine delivery mechanisms known to those of skill in the art. It is also understood that the antigen-growth factor-containing compositions of the present invention can further include adjuvants, preservatives, diluents, emulsifiers, stabilizers, and other components known and used in prior art vaccines. Should. Any adjuvant system known in the art can be used in the compositions of the present invention. Such adjuvants include Freund's incomplete adjuvant, Freund's complete adjuvant, polydisperse β- (1
, 4) bound acetylated mannan ("Acemannan"), TITERMAX® (a polyoxyethylene-polyoxypropylene copolymer adjuvant obtained from CytRx Corporation), Chiron Corpora.
Modified Lipid Adjuvant from Cambridge Biotech (Cambri)
dge Biotech), including adjuvants derived from saponins, killed pertussis, lipopolysaccharide (LPS) from Gram-negative bacteria, large polymer anions such as dextran sulfate and inorganic gels such as alum, aluminum hydroxide or aluminum phosphate. But not limited to these.

Carrier proteins that can be used in the antigen-containing growth factor-containing composition of the present invention include maltose binding protein “MBP”; bovine serum albumin “BSA”; keyhole limpet hemocyanin “KLH”; ovalbumin; flagellin; Thyroglobulin; serum albumin of any species; gamma-globulin of any species; syngeneic cells;
a syngeneic cells having the a antigen; and polymers of D- and / or L-amino acids.

Further, the term “effective amount” refers to an amount of a growth factor that, when administered to a human or animal, elicits an immune response, prevents cancer, reduces cancer, or inhibits the spread and growth of cancer. Say This effective amount is readily determined by one skilled in the art according to routine methods.

For example, the immunogenic growth factor composition of the present invention generally comprises between 1.0 μg and
The parenteral or oral administration can be in the range of 1.0 mg, but this range is not intended to be limiting. The actual amount of the growth factor composition required to elicit an immune response will vary depending on the immunogenicity of the growth factor composition being administered, the condition being treated and the immune response of the individual patient. Will vary for each patient. Accordingly, the particular amount administered to an individual will be determined by routine experimentation and based on the training and experience of those skilled in the art.

The growth factor-containing compositions of the invention are used to produce antibodies directed to the portion of the growth factor that becomes immunogenic by presentation in a carrier. A composition comprising a growth factor in combination with a carrier can be administered directly to a patient to elicit an immune response. Alternatively, an anti-growth factor antibody is administered to an individual to passively immunize the individual against a growth factor and thereby prevent the onset of cancer growth, reduce existing cancer, or inhibit cancer growth. be able to.

In one embodiment, the invention encompasses a growth factor inserted into a carrier, such as a membrane carrier, such that a portion of the growth factor is presented on the surface of the carrier. Growth factors are usually not immunogenic because they are recognized as "self" by the immune system. However, when a growth factor is introduced into a host system in a carrier, for example by inserting a portion of the growth factor into the liposome surface, the presentation of the growth factor to the immune system is altered and the growth factor becomes an immunogen.

[0077] Immunogenic growth factor-containing liposomes can be made by reconstituting liposomes in the presence of purified or partially purified growth factors. In addition, growth factor peptide fragments can be reconstituted into liposomes. The invention also includes growth factors and growth factor peptide fragments that have been modified to increase antigenicity. For example, an antigenic moiety or adjuvant can be bound to or mixed with a growth factor. Examples of antigenic moieties and adjuvants include, but are not limited to, lipophilic muramyl dipeptide derivatives, non-ionic block polymers, aluminum hydroxide or aluminum phosphate adjuvants, and mixtures thereof.

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

[0079] Immunogenic compositions containing growth factors, modified growth factors and peptide fragments thereof are administered to humans or animals to induce immunity to these growth factors. The immunized human or animal expresses circulating antibodies to the growth factor that bind to the growth factor and thereby reduce or inactivate the growth factor's ability to stimulate the growth of cancer cells.

Liposomes with growth factors inserted into the membrane, as well as other immunogenic compositions containing growth factors, have also been used to obtain panels of monoclonal or polyclonal antibodies specific for growth factors. You. Antibodies are made by methods well known to those of ordinary skill in the art. Anti-growth factor antibodies bind to growth factors when administered to an individual and reduce the effective circulating concentration of growth factors. As a result, growth factor dependent growth of the cancer is prevented, reduced or inhibited.

The growth factor-containing compositions and anti-growth factor antibodies of the invention are administered to a human or animal by any suitable means, preferably by injection. For example, growth factors reconstituted in liposomes are administered by subcutaneous injection. Whether produced internally or provided by an external source, circulating anti-growth factor antibodies bind to the growth factor and reduce or inactivate the growth factor's ability to stimulate the growth of cancer cells.

[0082] Liposomes that can be used in the compositions of the present invention include those known to those of skill in the art. Any of the widely used lipids useful for making liposomes can be used. Standard bilayer and multilamellar liposomes can be used to produce the compositions of the present invention. Although any method of producing liposomes known to those skilled in the art can be used, the most preferred liposomes are described in Alving et al., Infect. Immun.
60: 2438-2444, 1992, which is incorporated herein by reference. Liposomes can optionally contain an adjuvant. Preferred adjuvants are detoxified lipid A, for example monophosphoryl or diphosphoryl lipid A.

When the vesicle is a liposome, the growth factor generally has a hydrophobic tail that inserts into the liposome membrane when it is formed. In addition, the growth factor can be modified to include a hydrophobic tail so that the growth factor can be inserted into the liposome. For example, a growth factor gene is fused to an oligonucleotide sequence encoding a hydrophobic tail. This modified gene is inserted into an expression system and expressed using methods known in the art to obtain a growth factor fusion protein with a hydrophobic tail. Alternatively, growth factors are exposed to the surface of preformed liposomes by chemical bonding or electrical insertion.

When the vesicle is a baculovirus-derived vesicle, the recombinant growth factor is expressed on the insect cell membrane as a natural result of processing by infected insect host cells. As in the liposome embodiment described above, the growth factor can be modified to include a hydrophobic moiety in the recombinantly expressed protein to facilitate insertion into the vesicle membrane. Growth Factor Antibodies The antibodies provided herein are monoclonal or polyclonal antibodies having binding specificity for growth factors, such as bFGF and VEGF, and their antigenic peptides and fragments. Preferred antigenic fragments include a peptide having the amino acid sequence set forth in SEQ ID NOs: 1-9. The most preferred antibodies are monoclonal antibodies because of their higher specificity for the antigen. These antibodies are antigen-specific and show minimal or no cross-reactivity with other growth factor proteins or peptides.

The monoclonal antibodies of the invention are produced by immunizing an animal, such as a mouse or rabbit, with a growth factor (eg, bFGF) or an immunogenic fragment thereof (eg, a heparin binding domain peptide of bFGF). Splenocytes are collected from the immunized animals and hybridomas are produced by fusing the sensitized splenocytes with a myeloma cell line, such as mouse SP2 / O myeloma cells (ATCC, Manassas, VA). These cells induce fusion by adding polyethylene glycol. Hybridomas are seeded chemically on a selective medium (HAT) containing hypoxanthine, aminopterin and thymidine.

Subsequently, the hybridomas are screened for the ability to produce monoclonal antibodies to specific growth factors or related proteins and fragments. Growth factors and related proteins and peptides used for screening purposes are obtained from the analyzed samples. Alternatively, the desired growth factor can include a recombinant peptide produced according to methods known to those skilled in the art. Hybridomas producing antibodies that bind to growth factors or their protein fragments are
Cloned, expanded and stored frozen for future production. Preferred hybridomas produce monoclonal antibodies having the IgG isotype, more preferably the IgG1 isotype.

[0087] Polyclonal antibodies are produced by immunizing an animal, such as a mouse or rabbit, with a growth factor or a protein fragment thereof as described above. Subsequently, serum is collected from the animal and the antibodies in the serum are screened for binding reactivity to an antigen reactive with the growth factor or protein fragment, preferably the monoclonal antibody described above.

Peptide or Protein Fragments Growth factors, peptides or fragments thereof containing an immunogenic region can be produced from the proteins described above and immunogenic activity using techniques and methods known to those skilled in the art. Can be tested for For example, full-length recombinant bFGF (rbFGF) can be produced using a baculovirus gene expression system. Full-length proteins are described, for example, in Enjyoji et al. (Biochemistry 34: 5
725-5735 (1995)), can be used to cleave or digest into individual domains using various methods. RbFGF is treated with a digestive enzyme, human neutrophil elastase, according to the method of Engeorg et al., And the digest is purified using a heparin column. Human neutrophil elastase is b
Cleavage FGF into several fragments: one contains the heparin binding domain, and the other contains the receptor binding domain. To obtain additional fragments, the protein is preferably Balian et al. (Biochemistry 11:
3798-3806 (1972)), treating with a digestion compound, hydroxylamine, and purifying the digest using a heparin column.

Alternatively, fragments are produced by digesting the entire protein or a large fragment thereof exhibiting immunogenic activity, and then removing one amino acid. Each progressively shorter fragment is then tested for immunogenic activity.
Similarly, fragments of various lengths can be synthesized and tested for immunogenic activity. By increasing or decreasing the length of the fragment, one of ordinary skill in the art will be able to use routine digestion, synthesis and screening methods known to those of skill in the art to make amino acids within proteins required for immunogenic activity. The exact number, identity and sequence of can be determined.

The desired peptides and peptide fragments can be obtained from Infinity Biotech.
Research and Resources (Infinity Biotech Research and Resources)
(Upland, PA) or Research Genetics (Research)
Genetics) (Huntsville, Ala.).

As the recognition that the progressive growth and expression of primary tumors and metastatic lesions is dependent on angiogenesis has increased, considerable effort has focused on the potential therapeutic manipulation of the angiogenic compartment of tumors. In this regard, a rationale has been developed for targeting a specific immune response to the appropriate epitope on the FGF molecule to endogenously control tumor growth. Peptides derived from functional domains of FGF were screened for their ability to competitively inhibit FGF-stimulated endothelial cell proliferation. Peptides that blocked endothelial cell growth but did not block tumor cell growth in vitro were selected as target antigens, covalently linked to limpid vesicles containing lipid A as adjuvant, and stimulated angiogenic stimulation. Factor, FGF
Used to vaccinate mice in an attempt to generate a specific immune response directed at Using this liposome technology, the inventors of the present invention have shown that vaccination of mice with the heparin binding domain but not the receptor binding domain results in inhibition of angiogenesis in vivo, and the generation of two mouse tumors in an experimental metastatic model. It proved that proliferation and expression were significantly reduced. Therefore, by inhibiting positive regulators of angiogenesis, reversal of the angiogenic phenotype of malignant cancers could be achieved.

Molecular characterization of basic and acidic fibroblast growth factor (FGF) has confirmed the existence of two classes closely related to angiogenic factors. Acidic and basic FGFs (hereinafter aFGF and bFGF) have been identified in a number of molecular forms that are the product of proteolytic processing at homologous sites. FGFs are associated with a number of physiological functions, such as reproduction, growth and development. Identification of specific functional domains in the primary structure of bFGF is described by Baird et al. (Proc. Natl. Acad. Sci. USA
85 (1988)), which is incorporated herein by reference.

[0093] Basic fibroblast growth factor (bFGF) is an important stimulator of angiogenesis involved in tumor progression. As shown in Examples 1-15, two synthetic peptides of bFGF, one derived from the heparin binding domain and the other derived from the receptor binding domain, are bFGF stimulatory in vitro. HUVEC proliferation was inhibited. C57BL / 6J mice were separately vaccinated with synthetic peptides covalently complexed with liposome vesicles containing lipid A.
Serum analysis revealed an antibody response in mice immunized with the heparin binding domain peptide, but not in mice vaccinated with control liposomes or with the receptor binding domain peptide conjugated to the liposome. To determine whether the effect of vaccination with bFGF stimulated a response in vivo, a gelatin sponge containing bFGF was implanted into the liver of vaccinated mice. Histological analysis of the sponge showed that mice vaccinated with the heparin binding domain peptide showed no sponge cell infiltration and absence of angiogenesis. Thus, a growth factor vaccine comprising an immunogenic peptide, particularly a heparin binding domain peptide of bFGF, can be used to stimulate an immune response that correlates with inhibition of a bFGF-stimulated in vivo response.

[0094] Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and is involved in processes including angiogenesis and vascular permeability. VEGF is a 40-45K homodimer and has, in a limited manner, an amino acid sequence similar to that of platelet-derived growth factor (Soker et al. Cell, 92: 735-745 (1998)). See, which is incorporated herein in its entirety). As shown in Example 14, V
A previously unidentified immunogenic fragment of EGF can be administered to elicit a vigorous immune response in the host, resulting in inhibition of tumor growth or reduction of cancer.

According to the novel findings disclosed herein, bFGF peptides and or VEGF
Growth factors such as peptides can be used in compositions suitable for treating hyperproliferative diseases such as angiogenesis-related diseases, and especially cancer.

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

Peptide A: YCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEE
GVVSIKGV (SEQ ID NO: 1) Peptide B: SNNYNTYRSRKYSSWYVALKR (SEQ ID NO: 2) Peptide C: CRTKPEKKCDKPRR (SEQ ID NO: 3) Peptide D: CECRPKKDRTKPEKCDKPRR (SEQ ID NO: 4) Peptide E: APTTEGEQRKFKRKFSKERKVCERKKRSKV
DKPRR (SEQ ID NO: 6) Peptide G: CNDEGLESVPTEESNITMQIMRIKPH (SEQ ID NO: 7) Peptide H: CNDEGLESVPPTEE (SEQ ID NO: 8) Peptide I: CEESNITMQIMRIKPH (SEQ ID NO: 9) Peptide J: YCKNGGFFLFLPDGRKDVGERPKVGERKVGEVKVGERKVKVGERPKPDG
RGVVSIKGV (SEQ ID NO: 10)

Formulations A naturally occurring or synthetic protein, peptide or protein fragment containing all or an active portion of an immunogenic protein or peptide is prepared in a physiologically acceptable formulation, eg, in a pharmaceutically acceptable carrier. It can be prepared using known techniques. For example, the protein, peptide or protein fragment can be combined with a pharmaceutically acceptable excipient to form a therapeutic composition.

Alternatively, the gene for a protein, peptide or protein fragment containing all or an active portion of an immunogenic peptide can be delivered in a vector for continuous administration using gene therapy techniques. The vector can be administered in a medium having specificity for a target site, eg, a tumor.

The compositions of the present invention can be administered in solid, liquid or aerosol form.
Examples of solid compositions include pills, creams and implantable dosage units. The pill can be administered orally. Therapeutic creams can be administered topically. Implantable dosage units can be administered locally, for example, at the site of a tumor, or can be implanted, for example, subcutaneously, to release the therapeutic composition systemically. Examples of liquid compositions include formulations adapted for intramuscular, subcutaneous, intravenous, intraarterial injection, and formulations for topical and ocular administration. Examples of aerosol formulations include inhalation formulations for administration to the lung.

[0101] The compositions of the present invention can be administered by standard routes of administration. Generally, the compositions of the present invention can be administered by a topical, oral, rectal, nasal or parenteral (eg, intravenous, subcutaneous or intramuscular) route. In addition, the compositions of the present invention can be incorporated into sustained release matrices, such as biodegradable polymers, and these polymers are implanted where delivery is desired, eg, near the site of a tumor.
The method includes administration of a single dose, administration of repeated doses at predetermined time intervals, and continuous administration for a predetermined period.

As used in the present invention, a sustained release matrix is a matrix made of a substance, usually a polymer, which can be degraded by enzymatic or acid / base hydrolysis or by dissolution. Once inserted into the body, the matrix is affected by enzymes and body fluids. The sustained release matrix is desirably a biodegradable material such as liposomes, polylactide (polylactide acid), polyglycolide (polymer of glycolic acid), polylactide coglycolide (copolymer of lactic acid and glycolic acid), polyanhydride, poly (ortho). Esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides,
Selected by nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinyl pyrrolidone and silicon. A preferred biodegradable matrix is a matrix of any one of polylactide, polyglycolide or polylactide coglycolide (copolymer of lactic acid and glycolic acid).

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

The compositions of the present invention can be administered in combination with other compositions and methods for treating a disease. For example, undesired cell proliferation can be routinely treated with surgery, radiation or chemotherapy in combination with the administration of the composition of the invention,
And subsequently, an additional dose of the composition of the present invention can be administered to the patient to stabilize and inhibit the growth of any remaining unwanted cell growth.

Diseases and Conditions to be Treated The methods and compositions described herein treat human and animal diseases and processes resulting from abnormal or unwanted cell proliferation, especially endothelial cell proliferation. And these diseases and processes include hemangiomas,
Solid tumor, leukemia, metastasis, telangiectasia psoriasis, scleroderma, suppurative granuloma, myocardial angiogenesis, plaque neovascularization, corneal disease, rubeosis, neovascular glaucoma, diabetic retinopathy, post lens fibrosis Disease, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcers, fractures, keloids, angiogenesis, hematopoiesis, ovulation, menstruation and placenta formation. The methods and compositions of the present invention are particularly useful for treating angiogenesis-related diseases and disorders by inhibiting angiogenesis.

The methods and compositions described herein are particularly useful for treating cancer, arthritis, macular degeneration and diabetic retinopathy. Administration of a composition of the present invention to a human or animal having a pro-angiogenic metastatic tumor is useful to prevent the growth or spread of such tumors.

The compositions and methods of the present invention are further illustrated by the following non-limiting examples, which should not be construed in any way as limiting the scope of the invention. On the contrary, after reading this description, various other embodiments, modifications and variations thereof may be suggested to one skilled in the art without departing from the spirit of the invention and / or the appended claims. It should be clearly understood that measures may be taken for equivalents.

EXAMPLES Example 1 Construction of Recombinant Baculovirus Containing Growth Factor Baculovirus expression vector was obtained from Webb, NR, etc. (1989) Proc. Natl. Acad.
Sci. USA 86, 7731-7735, which is incorporated herein by reference. Recombinant baculoviruses containing cDNA encoding full-length growth factor under the transcriptional control of the polyhedron promoter were used to convert recombinant pAc-growth factor DNA into wild-type Autographa californica by calcium phosphate precipitation. Nuclear polyhedrosis virus (AcMNPV) DNA and co-transfection
) To produce.

Example 2 Growth Factor Expression and Purification The closure-negative virus obtained from Example I was Spodoptera frugiperda (Spod
Plaque purified and grown in optera frugiperda 9 (Sf-9) cells. Infected Sf-9 cells were isolated from Webb, NR et al. (1989) Proc. Natl. Acad. Sci.
Grow in monolayer or suspension as described in SA 86, 7731-7735. Briefly, Sf9 cells were isolated from Summers, MD and Smith, (1987
) Baculovirus expression vector and insect cell culture procedure method manual (A Ma
nual of Methods for Baculovirus Expression Vectors and Insect Cell Cultu
re Procedures) (Texas Agricultural Experiment Station, College Station, TX), Bull. 1555, and cultured at 27 ° C. in TNMFH medium supplemented with 10% (v / v) heat-inactivated fetal calf serum. Extraction and purification of the recombinantly expressed growth factor is performed by standard methods.

Example 3 Preparation of Growth Factor-Containing Liposomes Purified growth factors are electro-inserted into liposomes by standard methods known in the art (Mouneime, Y. et al., 1990, Biochem. Or cited in the specification). Incorporation of growth factors or their fractions into liposomes can include the following: (a) Reconstitution of lyophilized liposomes of known lipid components in the presence of aqueous growth factor (or growth factor fragment) solution Hydration, thereby creating mono- or multi-lamellar liposomes containing growth factors contained in the liposome lumen or trapped in the aqueous layer between the lipid membranes of the multi-lamellar vesicles; (b) reconstituted Electro-insertion of the growth factor or a fraction thereof into the liposomes, in which case the growth factor will be present in the lumen or between the lipid membranes; (c) Preparation of a known composition with lyophilized fragments of the growth factor Reconstitution of the liposome, where each fragment binds to the liposome lipid bilayer so that the peptide fragment is exposed to the outer surface of the liposome. (D) conjugation of the active agent to the liposome surface; and (e) a growth factor, a fraction thereof, or a synthesis that mimics the activity of the composition or growth factor of the present invention. Any other reconstitution or active reagent combination method that results in the production of an immune response (fluid or cells) directed against the peptide.

Example 4 Preparation of Growth Factor-Containing Baculovirus Vesicles Growth factor-containing vesicles are produced as follows. Insect-derived vesicles containing the recombinant growth factor in the membrane are obtained using baculovirus-infected insect cells. More specifically, Spodoptera frugiperda IPLB-Sf21-AE clonal isolate 9 (designated Sf9) insect cells were cultured and cultured in Proc. Natl.
ad. Sci. USA 86, 7731-35 (1989), and U.S. Patent Nos. 4,745,051 and 4,879,236, both issued to Smith et al., which are incorporated herein by reference. Infected with a recombinant baculovirus containing cDNA encoding full-length growth factor as described more fully in US Pat. Approximately 0.8 × 10 ⁶ Sf9 cells were transferred to an Excel medium (J
Seed into a 1 liter Spinner flask containing RH Scientific, 95695 Woodland, CA. These cells were grown at 27 ° C, 50
Incubate in a% O ₂ atmosphere. 3.5-4.0 million cells / ml
When the density of baculovirus containing recombinant growth factor is reached,
Add to media at a multiplicity of 600 viruses / cell. Vesicle production begins approximately 24 hours after baculovirus infection. Peak vesicle formation is achieved approximately 72 hours after the initial baculovirus infection. Collect the contents of the flask, and
Centrifuge at pm to remove cells and cell debris. Collecting the supernatant containing the vesicles,
It is then centrifuged at 20,000 RPM for 30 minutes on 50% Percoll containing 0.1 M sodium bicarbonate pH 8.3 using an angle fixed rotor. A double band below the interface between Percoll and cell culture medium was collected, and the suspension was subjected to 20,000 RPs in a swing-bucket rotor.
Centrifuge at M for 30 minutes. Two bands with a density of 1.05 g / ml for vesicles and 1.06 g / ml for baculovirus particles can be observed at the top and bottom of the slope. These vesicles have growth factors displayed on their outer surface and can be used for immunization. Vesicles are 20,000RP
Wash three times with 0.1 M sodium bicarbonate pH 8.3 using a 20 min centrifugation at M and resuspend in this same buffer.

Example 5 Immunization with Growth Factor-Containing Compositions Immunogenic growth factor-containing compositions containing liposomes or baculovirus-derived vesicles are injected into humans or animals at a dose of 1-5000 μg / kg body weight. . Is the antibody titer against a growth factor determined by ELISA using recombinant protein and horseradish peroxidase-conjugated goat anti-human or animal immunoglobulin, or by other serological techniques (sandwich ELISA), or does it currently exist? Or biological activity assays such as those specifically developed for individual growth factors (eg, natural or synthetic cytokine or growth factor detoxification or competition assays). Booster infusions are administered as needed to achieve protective antibody levels sufficient to reduce or detoxify growth factor activity in vivo. Detoxification 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 a high titer of anti-growth factor antibody has been achieved, the IgG fraction is isolated from the blood and used to passively immunize the individual as described in Example 7 below.

Example 7 Passive Immunization The anti-growth factor antibodies isolated from the species to be passively immunized were approximately 0.1 mg / kg body weight.
Administer intravenously as a dose value of 5-50 mg. The dose and frequency of administration will be determined in experimental animals challenged with various tumor types, and will be tailored to the particular type of tumor being treated and the particular individual. Important considerations are tumor aggressiveness, propensity for metastatic spread, target organs for metastasis, availability of tissue access by angiogenesis / antibodies of target organs, and stage of tumor development. While it will be true that standard treatment modalities can be universally protected, it is also true that effective treatments will only be achieved with criteria that are individualized based on tumor type.

Example 8 Preparation of peptide-conjugated liposomes as an anti-bFGF vaccine The following method and protocol were used to prepare an anti-growth factor vaccine, especially an anti-bFGF vaccine.

Peptide Selection The peptides selected for use in the present invention can include whole growth factor proteins or immunogenic fragments thereof. In this example, the immunogenic peptide was selected from the bFGF amino acid sequence. Incorporation of FGF peptide into liposomes dimyristoyl-glycero-3- [phospho-lac- (1-glycerol)] (
DMPG), dimyristoyl-glycero-3-phosphochlorin (DMPC), cholesterol (Avanti Polar Lipids, Alabaster, Ala.), Pyridyl dithiocholesterol (PDS-Chol) and lipid A (List Biological Labora)
tories, Inc., Campbell, Calif.)
It contained 1 ml of lipid A and was prepared in a molar ratio of 1: 9: 7.5: 1. The lipid was mixed in a round bottom flask and the solvent was removed by rotary evaporation. Multilamellar vesicles are prepared by hydrating dried lipids in 6.4 ml of sterile water (BioWhittaker, Walkersville, MD) containing 2 mg of either the heparin binding domain peptide or the receptor binding domain peptide of FGF-2. Prepared. The liposome formulation is incubated overnight at room temperature (RT) with gentle orbital shaking,
Lyophilized and resuspended in 2.135 ml 0.1 M citrate phosphate buffer pH 5.6. 200 microliters of each peptide was added to each liposome preparation, followed by incubation for 16 hours at RT on an orbital shaker. 1 liposome
Centrifugation was performed at 5,000 rpm at 4 ° C. for 30 mm. For injection, liposomes are
The suspension was resuspended in non-pyrogenic, Mg ^{2+ -free} and Ca ²⁺ -free PBS to a final volume of 5 ml. Quantification of liposome-associated peptides was performed by amino acid analysis (MA BioServices, Rockville, MD). Groups-a total of eight groups for initial immunization-three groups immunized with a composition containing liposomes with complexed peptides-immunized with a composition containing liposomes with passively encapsulated peptides Three groups-liposomes with buffer only, one group using lipids instead of complex liposomes-liposomes with buffer only, one group using lipids from those that are passively encapsulated- All of these groups contain 22 μg lipid A (c) lipid A / μmol phospholipid.

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

Certain pathogen-free C57BL / 6J male mice, 5-7 weeks of age, were purchased from The Jackson Laboratory (Bar Harbor, Maine). Mice were housed in a specific pathogen-free facility. In conducting the studies in this report, researchers should provide guidance on the care of laboratory animals and laboratory animal resource facilities, the National Academy of Sciences, and the Guide for Laboratory Animals and C
are of the Institute of Laboratory Animal Resources, National Academy of
Sciences, National Research Council). A population of mice was isolated at two-week intervals using liposomes containing either heparin binding domain peptide (L (HBD)) or receptor binding domain peptide (L (RBD)) (0.1 ml, intramuscular). Three treatments were performed. Each liposome injection delivered 1 μmol phospholipid, 200 μg lipid A and 30 μg peptide. The control group contained liposomes without complex peptide (L (LA)) and PBS. Thirteen days after the last injection, mice were bled from the lateral tail vein and serum was collected for antibody analysis. The next day, the mice were challenged intravenously with tumor cells or
Sponge (liver transplant) filled with GF-2 or B16BL6 was challenged.

Liposomes MLV for conjugated peptides with the following molar ratios: 9: 1: 7.4: 2: 0.02 DMPC: DMPG: CHOL: PDS-C
HOL: MLA (10 mol% PDS-CHOL) MLV for passive encapsulation 9: 1: 7.5: 0.02 DMPC: DMPG: CHOL: MLA

Dose The target dose generally contains 10-50 μg of peptide + 100 μg of MLA. Enough for three injections per mouse over the course.

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

Aliquots of lipids (per group), for a total of 8 groups)

[0123]

[Table 1]

MLA was added at 22 μg / μmol; List Biologicals, Inc. (Li
lot number 4013B obtained from St Biologicals, Inc.).推定 Estimated 100 μg MLA per injection, enough for about 50 injections,
Using 1.5 μg of total peptide at 10 μg per injection, estimated to be approximately 50% peptide encapsulation, sufficient for approximately 50 injections. # Dissolve 1 mg / vial of MLA in chloroform: methanol, 1: 1
Make approximately 1 ml / vial and pool.

Pyridyldithio- for surface-complexing cysteine-terminal peptides with liposomes
Preparation of pyridyldithiocholesterol (PDS-Chol) for surface conjugation of cysteine-terminal peptides to liposomes To make relatively non-immunogenic peptides more immunogenic, cysteine-terminal derived from VEGF amino acid sequence Surface complexation of peptide and liposome was performed. PDS-Chol is a thiol-reactive cholesterol analog that binds free thiols. The above-mentioned peptide is used in an aqueous liposome environment in PDS-
Has an amino-terminal or carboxyl-terminal thiol that binds to The peptide complexes with multilamellar vesicles (MLVs; liposomes) both inside and outside the MLV, thereby favoring a depo or sustained release effect with respect to antigen presentation.

The method used involves modifications such as Carlsson. Day 1 1.772 g (44 mmol) of thiocholesterol (Aldrich Chem.,
# 13, 611-5) was dissolved in 2.6 ml of chloroform. 13x100m
A glass screw cap tube was used and the composition was kept under nitrogen. The composition dissolved almost completely. The mixing line gave a slight milky white color. 2. 1.982 g (9.0 mmol) of 2 ', 2' dipyridyl disulfide P
DS) (Aldrithiol-2; Aldrich Chem., Milwaukee, WI) was placed in a 25 ml vaccine vial and 5.6 ml of chloroform: glacial acetic acid, 100: 1 (v: v) was added. A stir bar was added and the composition was mixed and dissolved (about 2 minutes). 3. Thiocholesterol was added dropwise to the vigorously mixed PDS at a rate of about 30 drops per minute. It turned yellow. 4. Immediately after thiocholesterol addition, the vial was purged with nitrogen, sealed, covered with aluminum foil, and mixed overnight at room temperature. Day 2 5. The vial was placed in a warm water bath and the solvent was distilled off with a stream of nitrogen. Dry to a viscous oil. Acetic acid was odor evident. 6. Using chloroform to wash out of the reaction vial, the composition was transferred to a 250 ml round bottom flask for recrystallization. 7. The composition was rotary evaporated to remove chloroform to give a clear yellow viscous oil. A stream of nitrogen was reapplied to remove residual solvent. 8. The composition was recrystallized by adding 50 ml of ethanol at about 56 ° C. Stir to dissolve. The sample was completely soluble. When left at room temperature (about 20 minutes), it turned into an emulsion. Placed on ice for about 1 hour. Large white crystals formed. 9. The crystallization was completed by leaving at −20 ° C. for 2 days. A large amount of crystallization product appeared. Large white / yellow crystals. Day 5 10. Filtered and collected by washing with ice-cold (dry ice) ethanol. Dry for about 4 hours and gain weight. The structure was confirmed by IR and NMR. 11. Hexane: ethyl acetate: diethyl ether: glacial acetic acid, 9: 1: 1: 0.
1, TLC at v / v.

[0127]

[Table 2]

Day 18 Two recrystallizations using 100 ml of ethanol at 63 ° C. It was left at room temperature for about 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

[0129]

[Table 3]

Stock solution: # 512.77 at 20 mM = 10.26 mg / 1 ml chloroform.

The weight versus absorbance assay is based on the DTT release of 2-thiopyridone, which absorbs strongly at 343 nm. The extinction coefficient is 7.0 / mM at 343 nm. 1. Three separate samples of PDS-cholesterol were weighed in 13 x 100 tubes. 2. PDS-Chol and 2-PDS (2-Aldrithiol-2 ™; Aldrich)
For 2.5 mM (1.282 g / ml) stock in ethanol. 3. PDS-chol is diluted in ethanol and 2-PDS is 0.1 M
Diluted to 0.125 mM in sodium bicarbonate. Molecular weight of PDS-Chol = 512.77. 4. Dithiothreitol at 50 mM (7.72 mg / ml) in 0.1 M sodium bicarbonate was prepared.

Results Analysis using functional group specific spray: Sterol spray: Ferric chloride spray: Specific for sterol: Positive for suspected PDS-cholesterol and thiocholesterol, negative for 2-PDS.
Iodine vapor: 90-95% purity (estimated). Surface Conjugation of Peptides: Method (Biosafety Cabinet Used for Operation) Day 1 Lipids were aliquoted into depyrogenated 100 ml round bottom flasks in the following order; lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol. 2. The bulk solvent was removed by rotary evaporation. The bath was at 42 ° C. The rotation of the flask was set at 160 rpm. About 2 seconds until bulk solvent evaporates
Start with a 2 mm Hg vacuum and then increase the vacuum to 25 mm Hg and 5
Minutes. Nitrogen was bubbled into the flask and stoppered until the flask was ready to dry. 3. The opening of the flask was covered with filter paper and dried under vacuum for 3 hours. 4. While drying the lipids, the sterile water was deoxygenated by blowing nitrogen through a stoppered sterile 5 ml pipette for 20 minutes. Also prepare 10 ml of 10 mM acetic acid,
Then, it was sterilized by filtration. Stock (HOAc) is 17.5N. Twenty microliters of stock HOAc was added to 35 ml of water to 10 mM. pH
Was 3.4. Deoxygenated by bubbling nitrogen and filter sterilized with a 0.22 micron syringe filter. 5. The peptide was completely dissolved in sterile 10 mM acetic acid to 5 mg / ml.
Peptide information is shown in the table below:

[0133]

[Table 4]

Soluble The white powder dissolved completely and almost immediately. 6. 0.2 ml (1 mg) of the 5 mg / ml peptide solution was added to 6.4 ml of deoxygenated DIW and mixed gently. This is a peptide hydration solution. 7. The peptide hydration solution was added to the dried lipid. The flask was purged with nitrogen and capped with a glass and sealed with parafilm. 8. The bath was sonicated to resuspend the lipids. A milky suspension was obtained. Stir for a moment. 9. Placed on orbital shaker, covered with foil and set at 3.25 and stirred overnight at room temperature. Day 2 10. The preparation was transferred to a sterile 20 ml vaccine vial and lyophilized for 3 days. Day 3 A 0.1 M citric acid / phosphate buffer, pH 5.6 (C / P buffer) was prepared: 1.921 g citric acid was dissolved in 100 ml DIW ("A"); 5.3
Dissolve 7 g of dibasic and odium phosphate-7-hydrate in 100 ml of DIW (CCB "); mix 21 ml of" A "with 29 ml of" B ";
Then add 50 ml DIW. Deoxygenate by blowing with nitrogen and filter sterilize. 12. 2. Add 135 ml of sterile C / P buffer to pre-form liposomes, purge the vial with nitrogen, and stir vigorously for 1-2 minutes. The suspension was relatively homogeneous. Lyophilization can be a limiting factor due to the large liquid: vial volume ratio. 13. Each peptide was completely dissolved in sterile degassed 10 mM acetic acid to give 10
mg / ml. The powder dissolved immediately and the solid dissolved completely in about 1 minute with stirring. 14. Add 0.2 ml (2 mg) of a 10 mg / ml peptide / HOAc solution to the preformed vesicles above, purge the vial with nitrogen, seal and cover with foil. 15. The orbital shaker was set to 6 and mixed overnight at room temperature. Day 4 16. First spin: vials were rinsed twice with 1 ml aliquots of PBS, and then successively with 0.5 ml of PBS for a total of 2.5 ml of sterile PBS for lipids. For use in transfer, a 1 ml plastic pipette is best. The total volume of the preparation was 5 ml. The volume of the lipid pellet was measured and was found to be 1.5 ml. Sterile PBS and autoclaved Qakridge tubes were used. 17． Centrifuged at 15,000 rpm in a SS-34 rotor at 40 ° C. for 30 minutes at Sorvall high-speed. 18. The supernatant was carefully removed with a pipette and stored in frown. The tube was labeled as the first spin. Total volume 5 ml; lipid volume 1.5 ml
Therefore, about 3.5 ml is water, so this about 3.5 ml should be used for calculations and the BCA assay uses each peptide stock as a standard. 19. Second spin: about 5 ml of PBS was added to the pellet and vortexed to completely resuspend the pellet. A total of 30 ml of PBS was added and mixed upside down. 20. Centrifuged at 15,000 rpm in a SS-34 rotor at 40 ° C. for 30 minutes at Sorball High Speed. 21. The supernatant was decanted into a 50 ml tube labeled as a second spin and stored frozen. The control pellet was slower in both groups. The peptide pellet was firm and the supernatant was clear. 22. About 1 ml of PBS was added to the pellet and the pellet was carefully resuspended until a homogeneous suspension was obtained. Transferred to a 5 ml sterile snap cap tube. The total suspension was made up to 5 ml using sufficient PBS. 23. Assay for phosphorus; assay for incorporation by BCA (use each peptide as its own standard). Use 10 mg / ml frozen peptide stock.

Injection optimal = 100 μl containing 100 μg lipid A and 10 μg peptide intraperitoneally. Method for simply encapsulating the peptide: Day 1 Lipids were aliquoted into depyrogenated 100 ml round bottom flasks in the following order: lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol. 2. The bulk solvent was removed by rotary evaporation. Starting with a vacuum of about 22 mm Hg until the bulk solvent evaporated, the vacuum was then increased to 25 mm and performed for 5 minutes. 3. Dry under vacuum for 2 hours. 4. The lipid was hydrated as follows; 4.5 ml of sterile water was added to the lipid and bath sonication suspended the lipid. A relatively homogeneous suspension was obtained. 2 at room temperature
Left for hours. 5. Transferred to a 10 ml vaccine vial. The rotary flask was continuously rinsed with 1 ml fractions of sterile water. The vials were sealed using sterile butyl-type rubber vaccine vial septa and parafilm. 6. The vials were frozen on dry ice for 30 minutes. 7. A # 20 sterile hypodermic needle was placed through the septum and the airflow was subtracted for lyophilization. 8. The vial was placed in a freeze dryer. Two days will be sufficient to freeze dry the lipids. Day 2 9. A concentrated peptide solution (10 mg / ml) was prepared in 10 mM HOAc.
0.5 ml is required for a total of 5 mg of peptide. Once dissolved, the peptide solution was diluted in 2 ml of room temperature sterile PBS. 10. Next, 4.5 ml of this peptide solution was added to the lyophilized lipid,
The vial was stirred vigorously for 2 minutes. This resulted in a final concentration of about 100 mM for phospholipids. 11. The vial was left encapsulating at 4 ° C. for about 24 hours. Day 3 12. The lipid was transferred to a 35 ml sterile Oak Ridge tube for centrifugation. A 1 ml pipette was used for this transfer. The vial was flushed with two 1 ml aliquots of cold PBS. The total volume of the preparation was 7 ml. Pellet capacity is about 1.5
ml. 13. Centrifuged at 15,000 rpm in an SS-34 rotor at 4 ° C. for 30 minutes at Sorball High Speed. 14． The supernatant was removed by pipette and stored frozen as for the first spin. 15. About 5 ml of PBS was added to the pellet and stirred to resuspend the pellet uniformly. PBS was added to make a total of 30 ml and centrifuged as above. 16. The pellet was resuspended using PBS and transferred to a 5 ml snap cap tube for a total of 5 ml. 17． Assay for phosphorus and peptide encapsulation.

Example 9 In Vivo Testing to Demonstrate the Effectiveness of bFGF Heparin Binding Domain Peptide Vaccination For the Ability of Two Peptides Derived from the Functional Domain of the bFGF Molecule to Block bFGF-Stimulated Endothelial Cell Proliferation In Vivo analyzed. Peptide A is b
44 amino acid peptide corresponding to the heparin binding domain of FGF, sequence: YCKNGGFFLRIHPGDGRVDGVREKSDPHHIKLQLQA
EEGVVSIKGV (SEQ ID NO: 1).

Peptide B is a 21 amino acid peptide and corresponds to the receptor binding domain and has the sequence SNNYNTYRSRKYSSSWYVALKR (SEQ ID NO: 2)
have.

Peptides A and B were obtained from Infinity Technology, Inc. (Infinity Tec
hnology, Inc. (Upland, PA) and was hydrated in 10 mM acetic acid to a concentration of 10 mg / ml for use in proliferation assays and liposome formulations.

[0139] Peptides A and B were both incorporated into Lipid A containing liposomes and used separately in vaccination protocols. The peptide was conjugated to the liposome according to the method and protocol described above in Example 8. Mice were immunized and boosted twice, bled and sera analyzed by ELISA for anti-peptide antibody titers and cross-reactivity with bFGF.

A comparison of the titer and cross-reactivity of serum obtained from mice vaccinated with the liposomal heparin binding domain peptide of the whole bFGF or bFGF vaccine is shown in FIG. 4 and the liposome receptor binding domain of the whole bFGF or bFGF vaccine A comparison of serum titers and cross-reactivity from mice vaccinated with the peptide is shown in FIG.

Following the vaccination protocol, mice were challenged with bFGF-filled sponges and angiogenesis was assessed 14 days later, and these results are shown in FIG.

Gel Foam Sponge Model of Angiogenesis Gel foam sponge (Upjohn, Kalamazoo, MI) was used as described previously by Watanabe et al., Am. J. Path., 150: 1869-1880. Cut into 5 mm squares and hydrated in PBS at room temperature for 24 hours. After hydration, the sponge was dried on sterile gauze and dried, and FGF-2
Rehydrated in 80 μl of a solution containing [8 ng / ml] and heparin [10 U / ml]. The gel foam sponge was incubated at 37 ° C. for 1 hour before transplantation into mouse liver.

Mice were anesthetized with metafane and a small incision was made in the ventral area of the mouse to expose the liver. The gel foam sponge was fixed on the left lobe of the liver using cyanoacrylate glue, and the wound was closed with surgical staples. Fourteen days after transplantation, the mice were sacrificed, the sponges were removed from the liver,
And fixed in 10% buffered formalin for histological analysis.

Experimental Lung Metastasis Model Exponentially growing B16BL6 tumor cells were treated with short-term trypsinized (0.25% DI
FCO trypsin and 0.02% EDTA, 1 minute at 37 ° C.), wash and wash in Ca ²⁺ , Mg ²⁺ free PBS to 2.5 × 10 ⁵ cells / ml. Again. Mice were injected with 0.2 ml of the cell suspension (5 × 10 ⁴ tumor cells / mouse) through the lateral tail vein using a 27 gauge needle. Fourteen days after inoculation, the mice were sacrificed and the lungs were removed and fixed in formalin. Surface lung metastases were counted using a dissecting microscope (Olympus, 3x).

Exponentially growing LLC-LM tumor cells are harvested using a cell scraper, washed and made up to 1 × 10 ⁶ cells / ml in Ca ²⁺ , Mg ²⁺ free PBS. Resuspended again. Mice were injected with 0.2 ml of the cell suspension through the lateral tail vein using a 27 gauge needle. Seventeen days after inoculation, mice were sacrificed and lungs were removed, weighed, and fixed in 10% buffered formalin.

Histological Analysis Lungs and gel foam sponges were fixed in 10% buffered formalin. Tissues are routinely embedded in paraffin, sectioned at 6 μm, and
Histology Loves, Inc. Stained with hematoxylin and eosin (H & E) by (Molecular Histology Labs, Inc.) (Gaithersburg, MD). Micrographs were obtained using an Olympus 1X70 microscope.

Statistical Analysis The results were analyzed for statistical significance using a two tailed Student's t-test.

To determine whether the production of antibodies to bFGF molecules has an effect on bFGF-induced angiogenesis, a liposomal lipid A control, liposomes containing heparin binding domain peptide, containing receptor binding domain peptide After vaccination with liposomes or PBS, the recombinant human bFGF-containing gelatin sponge was implanted into the left lobe of the mouse liver. The sponge was removed 14 days after implantation. The vascular distribution and leukocyte infiltration were evaluated histologically. Well-defined blood vessels containing erythrocytes were found in sponges removed from mice vaccinated with control preparations (liposomal lipid A and PBS) and sponges removed from mice vaccinated with liposomes containing the receptor binding domain peptide of bFGF. (Figure. In addition, there was also a large amount of infiltration of leukocytes and fibroblasts. Sponge removed from mice inoculated with the receptor binding domain looked similar to the control. Histological analysis of sponges removed from mice vaccinated with heparin binding domain peptides revealed a lack of cell invasion and angiogenesis induced by bFGF.
Red blood cells were present in histological sections of sponges taken from mice vaccinated with the heparin binding domain peptide, but they lacked well-defined structures surrounding these red blood cells.

Delivery of HBD and RBD in Liposomal Vesicles: Induction of Antibody Response Peptides derived from the two functional domains of FGF-2, HBD and RBD,
Covalently complexed and encapsulated in liposome vesicles containing the potent adjuvant lipid A, these are treated every 6 weeks with 6
Used for a week. Each inoculation delivered 1 μmol phospholipids, 200 μg lipid A and 30 μg peptide. After treatment, mice are bled and native FGF-
2. Sera were pooled for analysis of serum immunoreactivity to HBD and RBD peptides in an ELISA format. Immunoreactivity is determined by the HGF of FGF-2.
Measured in mice treated with a liposome formulation containing BD peptide (L (HBD)). This antibody reacted against both the HBD peptide and the parent FGF-2 molecule, with endpoint titers of 1: 10,000 and 1: 5,000, respectively (FIG. 20).
. In contrast, serum collected from mice treated with RBD peptide conjugated to liposomes (L (RBD)) or mice lacking liposomes lacking the conjugated peptide (L (LA)) contains the receptor binding domain peptide, heparin It showed no immunoreactivity to the binding domain peptide or to the intact FGF-2 molecule. As expected, all mice treated with liposome preparations containing lipid A adjuvant developed antibody titers to lipid A (end point dilution of serum: 1: 30,00
0) (data not shown).

To evaluate the specificity of antibodies produced in response to L (HBD) inoculation,
Serum from the treated animals is used to separate another heparin binding molecule: vascular endothelial growth factor
GF) was evaluated for immunoreactivity. When assayed in an ELISA format, sera from L (HBD) treated mice did not bind VEGF (FIG. 20).

Inhibition of B16-BL6-induced angiogenesis and tumor growth in liver sponge grafts To determine whether vaccination can inhibit tumor-induced angiogenesis, a gelatin sponge containing B16BL6 melanoma cells was , A control liposome (liposome lipid A), a liposome conjugated to a heparin binding domain peptide, a liposome conjugated to a receptor binding domain peptide, or PBS, and then transplanted into the liver of a mouse. Fourteen days after implantation, sponges were removed and vascularized and leukocyte infiltration was examined histologically. Sponge removed from mice vaccinated with liposomal lipid A and PBS contained viable and growing B16BL6 tumor cells. In addition, angiogenesis, a well-defined blood vessel containing red blood cells, was evident in these sponges. Furthermore,
There was extensive cell infiltration of leukocytes and fibroblasts into these sponges. In contrast, histological analysis of sponges removed from mice treated with liposomes containing FGF heparin binding domain peptide revealed reduced cell infiltration and lack of angiogenesis induced by FGF (FIG. 3d) ). On the other hand, red blood cells were present on histological sections of sponges taken from mice treated with liposomes containing the FGF heparin binding domain peptide. Similar effects were seen with sponges removed from mice vaccinated with liposomes containing the receptor binding domain. Vaccination with heparin binding domain peptides blocked angiogenesis and did not allow tumor growth or development in the sponge. b
Similar to the findings obtained from sponges containing FGF, there is a very large amount of erythrocytes lacking distinct structures surrounding the erythrocytes, lacking well-defined capillary structures and endothelial cells Notably shortage.

The results of these experiments are provided in FIGS.

Inhibition of BJ6BL6-induced angiogenesis and tumor growth in liver sponge grafts A gel sponge model of angiogenesis can also be used to assess tumor-induced angiogenesis. Gelatin sponges containing 5 × 10 ⁴ B16BL6 melanoma cells were implanted into the livers of mice vaccinated with L (LA), L (HBD), L (RBD) or PBS. Fourteen days after implantation, sponges were removed and examined histologically. Sponges removed from mice treated with L (LA), L (RBD) and PBS contained B16BL6 tumor cells (FIG. 2).
1a-c). Angiogenesis into these sponges was massive, as evidenced by endothelial cells forming distinct capillary structures containing red blood cells. In contrast, sponges obtained from mice treated with L (HBD) did not have overt neovascularization, and B16BL6 tumor cell growth was similarly inhibited.
Again, red blood cells were observed, but there were no vascular or endothelial structures surrounding the red blood cells.

Example 10 In Vivo Test Demonstrating the Effect of bFGF Heparin Binding Domain Peptide on Lung Metastasis in Mice To test the efficacy of bFGF peptide derived from heparin binding domain for cancer inhibition, a group of 10 mice was Vaccinated, boosted and B
16B16 mouse melanoma was challenged by the intravenous route. Peptides A and B described in Example 9 were conjugated to liposomes according to the methods and protocols described above and described in detail in Example 8.

Mice were vaccinated, boosted, and 14 days after challenge with B16B16 mouse melanoma, the animals were sacrificed and their lungs removed and the number of superficial melanotic tumor nodes counted. (FIG. 1A). FIG. 1A shows (a) mice vaccinated with the bFGF peptide obtained from the heparin-binding domain conjugated to the liposome, (b) mice vaccinated with the peptide obtained from the receptor-binding domain conjugated to the liposome, ( c) Mean number of lung metastases in mice vaccinated with hollow liposomes and (d) unvaccinated mice (PBS control). FIG. 2 shows lungs obtained from the control group (liposome lipid A, upper row) and lungs obtained from the vaccinated group (heparin binding domain peptide, lower row).
2 shows the difference in surface transition.

As evidenced by the results of this experiment and shown in FIG. 1A, bFGF
Administration of a composition comprising a heparin binding domain peptide of the present invention significantly reduces the average number of lung metastases and is therefore an effective antitumor composition.

Treatment with L (HBD) inhibited microscopic B16BL6 metastasis in lungs by 96% when compared to lungs of mice treated with L (LA), L (RBD) or PBS. Mice treated with either L (LA), L (RBD) or PBS had large melanotic lesions covering the entire surface of the lung. Histological analysis of lung tissue obtained from L (HBD) treated mice revealed only a small number of tumors, and these were less than 10 cell layers thick. Control lungs had multiple melanotic lesions with cell layer thickness greater than 10.

Confirmation of the antitumor effect was achieved in a second model of experimental metastatic disease. LLC-L
Seventeen days after intravenous challenge with M, two mice in the control group died due to progressive tumor burden, while the remaining control mice showed dyspnea and decreased mobility. All groups were sacrificed on day 17, lungs were removed and lung weights were measured to assess tumor burden. Mice receiving L (LA) had a significant tumor burden limited to the lungs, while mice treated with L (HBD) showed a significant decrease in tumor burden (
p <10 ⁵ ) (FIG. 1B).

Example 11 Effects of Growth Factors and Liposomal Vaccine Compositions on Tumor Volume and Size Compositions containing bFGF-liposomes can be used to monitor the effects of such compositions on tumor size. Was administered to tumor-bearing mice.

Materials and Methods A. Preparation of vaccines containing liposomes The liposomes used in these studies were prepared by Infect Immun 60 (6): 2 from Verma et al.
438-2444 (1992). This vaccine comprises lipid, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol (CHOL) and lipid A,
DMPC: DMPG: CH in a pyrogen-free rotary evaporator flask
It was prepared by combining OL: LA at a molar ratio of 9: 1: 7.5: 1 (mol: mol). The flask was dried under high vacuum for at least 2 hours. The lipid was hydrated in sterile water at room temperature for 2 hours. The hydration concentration used was 50 mM for phospholipids. These samples were then lyophilized. Recombinant human bFGF was encapsulated in liposomes by dissolving in PBS and combining with the lyophilized lipids described above. The mixture was agitated vigorously to ensure that the lipid was resuspended and incubated at 4 ° C. for 2 days. The amount of vaccine injected was determined by the amount of protein desired per dose. In mice, and maybe in humans,
Adjuvant lipid A is generally preferred because of the vigorous response. In these studies, the dose of adjuvant was 200 μl per injection per μmol total phospholipids.
g and the antigen dose was 10 μg bFGF per injection. The concentration of liposomes is shown as total phospholipids, ie, the sum of DMPG and DMPC; the final concentration of liposomes (vaccine) was 10 mM phospholipid. The maximum effective dose will vary and this is under study.

B. Immunization schedule 6-8 week old C57BL / 6J or Balb / cByJ mice were
On days 21 and 21, liposomes containing bFGF were injected intraperitoneally for immunization.
Serum was collected on days 0, 14, 21 and 35. Serum was analyzed by ELISA for anti-bFGF activity. The mice in the control group received uninjected mice (the mice were bled for titer) and mice inoculated with liposomes alone and bled similarly to obtain anti-bFGF titers. Included.

C. Vaccine efficacy analysis a. Antisera from vaccinated mice were tested for anti-bFGF activity by ELISA.
Analyzed in the A assay.

B. Two tumor models were used in the analysis of antitumor therapeutics. B16F10, C
Developed by M. O'Reilly, a highly tumorigenic melanoma and Children's Hospital that induces macroscopic lung tumors 14 days after intravenous inoculation of 10 ⁶ cells in 57BL / 6J mice Lewis Lung Cancer-Low Metastatic (LL
C-LM). In addition, the efficacy of the vaccination protocol as an anti-cancer therapy was evaluated in both allogeneic and syngeneic lines. The former are highly immunogenic tumors and the latter are non-immunogenic.

A group of vaccinated, unvaccinated and control liposomal (no bFGF) vaccinated mice were challenged intravenously with B16F10 and analyzed on day 14 for lung tumors. Numerous tumors, visible under a stereo microscope at 4 × magnification, were measured in each group.

The efficacy of the anti-bFGF vaccine was analyzed for both primary tumor growth and subsequent development of metastatic disease using the LLC-LM model. After 14 days of the end of the enhanced, mouse to 10 ⁶ LLC-LM (taken directly from the mice with a tumor)
Was challenged. Tumor volume was measured 14-21 days after inoculation and compared between the groups. At that time (14-21 days after tumor inoculation), the tumor was excised from one half of the group and the other half underwent sham operation. Fourteen days after surgery, mice were sacrificed, lungs were excised and weighed, and lung metastases were counted using a stereomicroscope.

Results bFGF-liposome-vaccinated and control liposome-vaccinated Ba
Preliminary data obtained from lb / cByJ mice indicate that the bFGF vaccine
Stimulated FGF antibody response. The anti-bFGF titers were 1: 12,800 to 1: 1, in vaccinated Balb / cByJ mice in two separate experiments.
The range was up to 30,000 (FIGS. 10A and 10B). Similarly, C57BL
/ 6J mice range from 1: 10,000 to 1: 50,0 measured in individual mice.
Response to vaccination with an anti-bFGF titer of 00 (Figure 13).

Immune Balb / cByJ mice were challenged with LLC-LM (allogeneic),
And the primary tumor expression was evaluated over 21 days. Control liposomes - mean tumor volume of the vaccinated mice were challenged similarly bFGF- liposome - as compared to 290 mm ³ after tumor transplantation 14 days of vaccinated mice was 830 mm ³ (Figure 14). In this experiment, T / C = 0.35 (T / C was the control vaccine (
Liposome only, no peptide) divided by the number of pulmonary nodules in the vaccinated group), and the tumor size was reduced by 65% compared to the control.

LLC- in vaccinated and non-vaccinated Balb / cByJ mice
In a second experiment using LM, on day 14 after tumor implantation, the mean tumor volume of control liposome-vaccinated mice was similar to bFGF-liposome-
229 mm ³ compared to 111 mm ^{3 of} vaccinated mice (FIG. 15).
. In this experiment, T / C = 0.48 and tumor size was reduced by 52%.

Balb / cByJ mice were allogeneic for LLC-LM, and tumor size was smaller in all groups than was observed in syngeneic C57BL / 6J mice. Thus, the next set of experiments was performed using vaccinated C57BL / 6J mice, using either (a) LLC-LM (primary tumor expression and metastatic disease assessed) or (b) B16F10 (measured metastatic disease). (Both are syngeneic models) were challenged.

As shown in FIG. 13, C57BL / 6J mice responded to the vaccination and produced bFGF antibodies. To evaluate the protective efficacy of induced bFGF antibodies in syngeneic tumor lines, vaccinated mice were challenged with LLC-LM and primary tumor growth and subsequent metastatic disease were assessed. In this syngeneic strain, circulating bFGF antibodies raised in response to vaccination and bFGF did not alter the growth of primary LLC-LM in C57BL / 6J mice.

Vaccinated mice were used to assess the extent of metastatic disease following primary tumor removal.
She underwent surgical resection of the primary tumor. In the LLC-LM model of metastatic disease, removal of the primary tumor results in rapid growth of metastatic tumors in the lungs and at the same time increases lung weight. Vaccination had no effect on metastatic development after primary tumor removal as assessed by lung weight measured 14 days after primary tumor removal.

Finally, C57BL / 6J mice were vaccinated and challenged with B16F10 by the intravenous route to evaluate vaccine efficacy in a syngeneic model of experimental metastasis. Although the anti-bFGF titer in vaccinated mice was 1: 16,000, the number of B16F10 translocations in the lungs of bFGF-liposome vaccinated mice was similar to that of mice vaccinated with control liposome preparations or control mice (PBS
Was inferior to the number of metastases in the lungs.

Conclusions The data obtained in this experiment indicate that a vaccine composition comprising a growth factor, especially a fibroblast growth factor, may be used to identify cancerous tumors, especially allogeneic cancer tumors or those that are highly immunogenic. Demonstrates that it can be used effectively to prevent or reduce it. As shown in both parts of this experiment, mice immunized with fibroblast growth factor incorporated in liposomes and challenged with allogeneic tumors were compared to controls immunized with a control vaccine. On average, tumor size was reduced by 65% and 52%.
In addition, antibody titers in mice that received the vaccine composition containing the growth factors were significantly higher than mice that did not receive these vaccines.

Example 12 Effect of Heparin Binding Domain Peptide of bFGF on the Growth of B16BL6 or HUVEC Tumor Cells In Vitro As demonstrated in Example 9, the heparin binding domain peptide of bFGF is capable of bFGF-stimulated HUVEC proliferation in vivo Showed an inhibitory effect on This same effect was not seen when incubated with tumor cells in vitro.

Cell Lines Human umbilical vein endothelial cells (HUVEC; Clonetics, Walkersville, MD) are endothelial cell growth media supplemented with 1% L-glutamine (Biowhittaker, Walkersville, MD) and bovine brain extract (EGM-2, Clonetics
Walkersville, Md.), Humid air, 10% CO ₂ , 75c
Maintained at 37 ° C. in m ² cell culture flasks.

The B16BL6 variant of B16-F10 melanoma was obtained from the National Cancer Institute-Central Repository (Frederick, MD). Tumor cells were 5% heat-inactivated fetal calf serum and L-glutamine (BioWhittak).
er, in DEME supplemented with Walkersville, MD) (BioWhittaker, MD), was at 37 ° C. in a humidified atmosphere of 5% CO _2/95% air grown as monolayer cultures. These cells were passaged at 80% confluency, and
Eight generations were used. B16BL6 is mycoplasma and the following pathogenic mouse viruses: reovirus type 3, mouse pneumonia virus, K virus, Tyler's encephalitis virus, Sendai virus, mouse microvirus, mouse adenovirus, mouse hepatitis virus, lymphocytic choroidal meninges. It was confirmed that they did not have the flame virus, ectomeria virus and lactate dehydrogenase virus.

The Lewis Lung Cancer-Low Metastatic (LLC-LM) cell line is a Dr. judah. Folkman (Children
's Hospital, Boston), and was confirmed not to have the same mycoplasma and pathogenic mouse virus as described above. LLC-LM was maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum and 1% L-glutamine.

Proliferation Assay HUVECs of 2-5 generations were washed with PBS and trypsin-versene (Bi
oWhittaker, Walkersville, MD) with a 0.05% solution. Cells were grown in endothelial cell basal medium (EBM-2, Clonetics) supplemented with 2% heat-inactivated fetal calf serum (Hyclone, Logan, UT) and 2 mM L-glutamine.
, 2.5 × 10 ⁴ cells / ml in Walkersville, Md., And seeded in 96-well (100 μl / well) culture plates (Costar, Cambridge, Mass.) And 5% CO 2. ₂ for 24 hours at 37 ° C. Cells were treated with FGF-2 peptide, 1, 10 and 100 μg / ml.
With either a 22 amino acid control peptide or medium alone for 20 mm and then 10 ng / ml human recombinant FGF-2 (R & D Systems,
(Minneapolis, MN) or medium alone for 72 hours. Cell proliferation was measured by BrdU incorporation using an ELISA assay (Boehringer Mannheim, Indianapolis, TN) according to the manufacturer's instructions.

A proliferation assay using B16BL6 or LLC-LM was performed as follows.
Cells 5% fetal calf serum, minimal essential medium Dulbecco's supplemented with 1% L-glutamine (DMEM, BioWhittaker, MD) in the 2 × 10 ⁴ cells /
and suspended in a 24-well (500 μl / well) culture plate (Cost).
ar, Cambridge, Mass.) and 4% at 37 ° C. in 5% CO _2.
Incubated for hours. FGF-2 peptide was added at 1, 10, 100 μg / ml and incubated for 20 minutes. After incubation, FGF-2
(10 ng / ml) was added to the wells for 48 hours. Cell growth is measured using a Coulter counter (
Coulter, Columbia, Md.).

In an alternative method, B16BL6 melanoma cells were plated in 24-well plates at 10
Plated at 2,000 cells / well and incubated overnight. Various concentrations of heparin-binding peptide were added to the wells, followed by medium containing 5 ng / ml bFGF. BFGF added in the absence of peptide was B16B
L6 growth was not increased over media controls. In addition, no significant effect on the growth of B16BL6 cells was observed when incubated with the heparin binding peptide and measured by cell number. These results are provided in FIG. This example provides a method useful for selecting peptides useful in the methods and compositions of the present invention.

Inhibition of Endothelial Cell Proliferation The inhibitory activity of the 44 amino acid peptide (SEQ ID NO: 1) corresponding to the heparin binding domain (HBD) of FGF-2 was further evaluated and additional functional domains of FGF-2, receptor binding For analysis of the domain (RBD), these peptides at a concentration of 1, 10 or 100 μg / ml in the HUVEC proliferation assay were 5 ng / ml.
Co-incubated with ml of FGF-2. Addition of FGF-2 alone stimulates HUVEC proliferation as compared to cells incubated without this growth factor. Both HBD and RBD peptides are capable of treating HUVEC F in a dose-dependent manner.
Inhibits GF-2-induced proliferation, with inhibitory concentrations (IC ₅₀ ) of 70 and 20 μg, respectively.
/ Ml (FIG. 19). A 22 amino acid control peptide corresponding to the identified region of human angiostatin had no inhibitory effect on FGF-2-induced proliferation of HUVEC.

Example 13 L in mice vaccinated with the heparin binding domain peptide of bFGF
Growth and Inhibition of LC-LM Metastasis The efficacy of vaccination of mice with the heparin binding domain peptide bFGF in a liposome format was determined using the LLC-LM experimental metastasis model.

Mice were vaccinated with the heparin binding domain peptide of bFGF in liposome format using the same vaccination strategy as the B16BL6 experiment described above (3 vaccinations at 2 week intervals). Two weeks after the final boost, mice are given L
LC-LM was challenged intravenously. On day 17, control (no treatment) mice began to die due to tumor burden in the lungs. All groups were sacrificed. Lungs were removed and analyzed based on lung weight. T / C was determined by subtracting normal lung weights from the treated and untreated groups. Vaccination with heparin binding domain peptides
When compared to controls, it inhibited growth and expression of LLC-LM experimental metastases by 94%. Since these lungs have been photographed, they will be evaluated histologically. As shown in the results of FIG. 11, the growth and expression of metastases was significantly inhibited in mice inoculated with heparin binding domain peptides.

Example 14 Immunogenicity of Vaccine Compositions Containing Growth Factors Determining Antibody Titers and Specificity The pooled sera was either L (HBD), L (RBD), L (LA) or PBS. From mice treated with Anti-FGF-2-specific antibodies were measured by enzyme-linked immunosorbent assay (ELISA) (ELISA). Briefly, 96-well immulon 4 plates (Dynotech, Chantilly, VA) were prepared using recombinant FGF-2, a heparin binding domain peptide of FGF-2, a receptor binding domain peptide of FGF-2, vascular endothelium. Any of the growth factors (VEGF) (5
0 μl; 2 μg / ml in carbonate buffer), incubated overnight at 4 ° C., and blocked with 3% milk and 0.05% Tween 20 in PBS (PBS / Tween 20). Plates were washed three times with PBS / Tween 20 and incubated with serum serial dilutions in PBS / Tween 20 for 1 hour. Plates were washed and biotin-conjugated goat anti-mouse immunoglobin (Ig
2.) Biotin-conjugated goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgG together with G and IgM (K & P) or to measure subclass responses.
And IgM for 1 hour at RT. After washing, the wells were incubated with horseradish peroxidase (HRP) -conjugated streptavidin. Optical density was read at 540 nm. Antibody titers are expressed as the mean O.D. of normal mouse serum. D. The O.D. D. It was defined as the reciprocal of the serum dilution giving the reading.

Example 15 In Vivo Testing to Demonstrate the Immunogenic Effect of VEGF Peptide in Mice This example is incorporated into liposomes that are immunogenic for growth factor peptides when administered to humans or animals. Or growth factor peptides, particularly vascular endothelial growth factor (VEGF) peptide fragments. Successful immunogenic responses have been obtained in mice when VEGF peptides have been incorporated into liposomes by either active peptide conjugation or passive encapsulation.

A. VEGF peptides Three different murine VEGF peptides, designated peptides CE, were incorporated into liposomes. Peptide C of SEQ ID NO: 3 is a carboxyl-terminal peptide consisting of residues 110-115, six amino acids of exon 8, and an amino-terminal cysteine for cross-linking purposes. Peptide D of SEQ ID NO: 4, also a carboxyl-terminal peptide, contains residues 102-115, six amino acids of exon 8, and an amino-terminal cysteine for cross-linking purposes. Peptide E of SEQ ID NO: 5
Is an amino-terminal peptide corresponding to residues 1-20 of VEGF, with the addition of a C-terminal cysteine for cross-linking purposes. The amino acid sequence of each peptide is provided below: Peptide C CRTKPEKCDKPRR (SEQ ID NO: 3) Peptide D CECRPKKDRTKPEKCDKPRR (SEQ ID NO: 4) Peptide E APTTEGEQKSHEVIKFMDVYC (SEQ ID NO: 5)

B. Preparation of vaccine using liposomes i. Active Peptide Conjugation One method used to incorporate growth factor peptides into liposomes is
It involves chemically coupling a peptide to an amino acid via a thiol group. In these studies, a cholesterol analog, PDS-CHOL, was used to create thiol-reactive liposomes. PDS-CHOL is White
Vaccin 12 (2): 1111-222 (1995).
Thiocholesterol (Aldrich Chem, Milwaukee, Wis. # 13,
611-5) (1.772 g) is dissolved in chloroform while in a separate vial PDS (2 ′, 2′-dipyridyl sulfide or Ald obtained from Aldrich Chem.).
rithiol-2) (1.982 g) was dissolved in a 100: 1 mixture of chloroform: glacial acetic acid. Thiocholesterol was added dropwise at a rate of about 30 drops per minute to the vigorously mixed PDS. The vial was then purged with nitrogen, sealed, covered with foil, and mixed overnight at room temperature. After distilling off the solvent using a nitrogen stream, the sample was recrystallized by adding 50 ml of ethanol at about 56 ° C., and the sample was placed on ice for 1 hour.
Then, it was left at −20 ° C. for 2 days. The sample was collected after filtration with ice-cold ethanol and dried. Purity was determined to be 90-95% using thin layer chromatography and iodine vapor.

Once PDS-CHOL is prepared once, it is combined with lipid, dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol (CHOL) and lipid A in a 100 ml round bottom flask 9: A molar ratio of 1: 7.4: 2: 0.02 (DMPC: DMPG: cholesterol: PDS: Chol: lipid A) (mol: mol) was obtained. The lipid mixture was rotary evaporated using nitrogen and dried. Except for the final PBS wash, all operations were performed anaerobically using degassed liquids. Next, the peptide was dissolved in 10 mM acetic acid to a concentration of 5 mg / ml, and the 1 mg equivalent was diluted in 6.4 ml of deionized water. The peptide hydration solution was added to the above dry liquid, resuspended by bath sonication, stirred briefly, and mixed on an orbital shaker at room temperature overnight. The mixture was then lyophilized. 2. Add 135 ml of citrate / phosphate buffer (0.1 M) and
The liposomes were preformed by stirring for 2 minutes. An additional peptide is 10 mg / m
Dissolve in sterile 10 mM acetic acid at a concentration of 1 l.
ml (2 mg) was added. The mixture was covered with foil and mixed on an orbital shaker at room temperature overnight. After washing twice with PBS, the liposomes were washed with BCA (Bicinchr).
oninic acid, Pierce Chemical Co., Rockford, Ill.)
Integration was analyzed (for a description of the assay used, see Bartlett et al.
Bio. Chem. 234: 466-68 (1959)). The results of the integration assay indicated that there was 68%, 74% and 66% conjugation of peptides C, D and E, respectively.

Ii. Passive Encapsulation Another method used for incorporation of peptides of growth factors such as VEGF was passive or simple encapsulation. Again, the lipids were aliquoted in a pyrogen-free 100 ml round bottom flask in the following order: lipid A, DMPC, DMPG and cholesterol in 1: 1 chloroform: methanol. These lipids were rotary evaporated to remove bulk solvent and then dried. To hydrate the lipids, 4.5 ml of sterile deionized water was added, the mixture was sonicated and left at room temperature for 2 hours. After transfer to a vaccine vial, the mixture was lyophilized. A peptide solution (2.5 mg / ml in room temperature PBS from a 10 mg / ml stock in acetic acid) was added to the lyophilized lipids above and the vials were stirred vigorously for 2 minutes. The final concentration for phospholipids is about 1
It was 00 mM. The vials were left at 4 ° C. for 24 hours to allow encapsulation, and then washed twice with PBS. Subsequent analysis of the encapsulation showed that there was 33%, 19% and 19% encapsulation of peptides C, D and E, respectively.

C. Vaccine Efficacy Analysis Five Balb / cByJ mice, 6-8 weeks of age, contain one of the three above peptides of VEGF incorporated by either active conjugation or passive encapsulation. Liposomes were injected intraperitoneally for immunization. Target dose is generally 10-50
μg peptide + 100 μg lipid A. Serum was collected on day 35 and analyzed by ELISA for anti-VEGF activity. As a control, mice that were not injected with liposomes were also bled to obtain anti-VEGF peptide titers.

Preliminary data obtained from Balb / cByJ mice vaccinated with VEGF-peptide-liposomes and control mice indicate that the VEGF vaccine was anti-VEGF
Stimulates the F antibody response. When these peptides were conjugated to liposomes, individual Balb / cByJ mice had anti-VEGF peptide titers greater than 1: 100,000 for peptides 2 and 3 and a 1: 25,000 force for peptide C. (Fig. 16 (a) to (c)). For peptides incorporated by simple encapsulation, individual Balb / cByJ mice responded with peptides D and E with anti-VEGF peptide titers ranging from 1: 12,500 to 1: 50,000 ( FIGS. 17A to 17C.

The above experiments show that a vaccine composition comprising a growth factor, specifically a peptide epitope of vascular endothelial growth factor, incorporated into liposomes, either encapsulated or complexed, is a significant antibody in vivo. It reasonably proves that it can elicit a response.

Example 16 Inhibition of Proliferation and Expression of B16BL6 Experimental Metastases in Mice Vaccinated with VEGF Peptide in Liposomal Format The neuropilin receptor binding domain of VEGF (peptide F, SEQ ID NO: 6)
) Was covalently complexed to liposome vesicles containing lipid A. Mice were vaccinated with the peptide F liposome preparation using the same vaccine format as the bFGF peptide vaccine. Two weeks after the final boost, mice were challenged intravenously with B16BL6. The mice were sacrificed 14 days after the antigen administration. Vaccination with the peptide F liposome formulation resulted in significant inhibition of the B16BL6 experimental metastasis when compared to the liposome lipid A control (T / C = 0.33). Heparin binding domain peptide bFGF covalently linked to liposomes was used as a positive control in this experiment. The result of this experiment is shown in FIG.
And 18.

Although the present invention has been described in particular detail with reference to the disclosed embodiments, it will be understood that many variations and modifications may be made within the spirit and scope of the invention.

[Brief description of the drawings]

FIG. 1A is a graph showing the results of experiments performed to demonstrate growth inhibition of experimental lung metastases. L (HBD) (bFGF peptide from the heparin binding domain bound to liposomes), L (RBD) (receptor binding domain of bFGF bound to liposomes), L (LA) (hollow liposomes) or PBS The treated mice were challenged intravenously with 5 × 10 ⁵ B16BL6 tumor cells. Mice were sacrificed 14 days after inoculation, lungs were removed, and surface lung metastases were counted using a dissecting microscope. Tumor growth was evident in all groups assayed. Mice treated with L (HBD) showed 96% inhibition of superficial lung metastasis. FIG. 1B is a graph showing the results of experiments performed to demonstrate inhibition of LLC-LM experimental metastasis. 5 × to mice pre-treated with L (HBD) or L (LA)
They were challenged intravenously with 10 ⁵ LLC-LM tumor cells. Mice were sacrificed 17 days after inoculation, lungs were removed and lung weights were evaluated. Mice treated with L (HBD) inhibited the expression of surface lung metastases by 9S%.

FIG. 2: Photograph comparing differences in surface B16BL6 metastasis in lungs from mice treated with liposomal lipid A ((control) top row) and mice treated with bFGF peptide derived from the heparin binding domain (bottom row). It is.

FIG. 3 is a photograph comparing the effect of vaccination with a peptide from the heparin binding domain of bFGF on lung B16BL6 proliferation and expression of control vaccination.

FIG. 4 shows the reactivity of sera obtained from mice vaccinated with heparin binding domain peptide of bFGF (open squares), heparin binding domain peptide bFGF (open diamonds) with total bFGF and normal mice It is a graph compared with serum (NMS) (open circle).

FIG. 5: Receptor binding domain peptide of bFGF having all bFGF (open squares)
FIG. 4 is a graph showing the reactivity of sera obtained from mice vaccinated with a receptor binding domain peptide (open diamonds) and NMS (open circles).

FIG. 6A shows histological analysis of gelatin sponges removed from a mouse vaccinated with a PBS control, an LLA control, a heparin binding domain peptide of bFGF and a receptor binding domain peptide of bFGF. . As further described in Example 9, to determine if vaccination against bFGF molecules has an effect on bFGF-induced angiogenesis, a liposomal lipid A control containing a heparin binding domain peptide was included. Liposomes, liposomes containing receptor binding domain peptides, or P
A gelatin sponge containing recombinant human bFGF was implanted into the left lobe of mouse liver after vaccination with BS. 6B-6D show the histological results of experiments performed to demonstrate inhibition of FGF-2-induced angiogenesis. Gelatin sponges containing FGF-2 were implanted into the liver of mice pre-treated with L (HBD), L (RBD) or L (LA). Fourteen days after implantation, sponges were removed and cut for histology. (FIGS. 6B and 6C) Hematoxylin and eosin (H & E) staining of sponges obtained from mice treated with L (LA) or L (RBD) revealed cell infiltration and angiogenesis. (D) H & E staining of sponges from mice treated with L (HBD) revealed a marked reduction in cell invasion and angiogenesis.

FIG. 7 shows histological analysis of gelatin sponges removed from mice vaccinated with PBS control, LLA control, liposomes containing the heparin binding domain peptide of bFGF, and liposomes containing the receptor binding domain peptide of bFGF. I have. As further described in Example 9, control liposomes (liposome lipid A), liposomes conjugated with heparin binding domain peptides, to determine if vaccination can inhibit tumor-induced angiogenesis, Gelatin sponges containing B16BL6 melanoma cells were implanted into mouse livers after vaccination with liposomes or PBS conjugated to receptor binding domain peptides.

FIG. 8 shows that bFGF stimulated HUVEC proliferation in the presence of bFGF peptide: control peptide (filled circle), heparin binding domain peptide of bFGF (
(Open circles) and the receptor binding domain peptide of bFGF (open squares).

FIG. 9 shows the reproducibility of the effect shown in FIG. 8: bFGF is a HUVEC in the presence of the bFGF peptide, the heparin binding domain peptide of bFGF (open circles) and the receptor binding domain peptide of bFGF (open squares) Stimulated proliferation.

FIG. 10. Heparin-binding domain peptide of bFGF (HEP) or receptor-binding domain peptide of bFGF (BEPGF) on B16BL6 tumor cell growth in vitro
9 is a bar graph showing the comparative effect of REC).

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

FIGS. 12 (a) and 12 (b) provide graphs showing anti-bFGF titers in serum obtained from mice vaccinated with bFGF-liposomes or control liposomes. The data presented in these graphs represents the reactivity of sera obtained from groups of 5 vaccinated mice, and the sera were collected on day 35, pooled, serially diluted, and removed from ELISA ( The reactivity to total bFGF is analyzed by measuring the absorbance in an ELISA) assay.

FIG. 13 is a graph showing anti-bFGF titers in serum obtained from individual mice vaccinated with bFGF-liposomes. As discussed in Example 11, C
57BI / 6J mice were given bFG according to the protocol outlined in the Methods section.
Liposomes containing F were vaccinated. Serum from individual mice labeled B1-B5 was collected on day 35, serially diluted, and analyzed for reactivity to total bFGF by measuring absorbance in an ELISA assay. The reactivity of sera obtained from mice vaccinated with the liposome control was not different from NMS. The data shown correspond to the method and protocol of Example 11 as follows: NMS (open squares with dots), mouse 1 (closed diamonds), mouse 2 (closed squares with white dots), mouse 3 (Open diamonds); mouse 4 (closed square) and mouse 5 (open square).

FIG. 14 is a graph showing the size of primary tumors in mice vaccinated with bFGF or control liposomes 14 days after antigen administration. As discussed in Example 11, Balb / cByJ mice were vaccinated with liposomes containing bFGF (bFGF) or liposomes without bFGF (control) according to the protocol outlined in the Methods section. . On day 35, 1 × 1 subcutaneously on the back of the mouse
0 ^six of LLC-LM were challenged. The data shown represent the tumor volume measured 14 days after challenge for each of the 5 mice in each vaccination group.

FIG. 15 is a bar graph showing primary tumor size in mice vaccinated with bFGF or control liposomes 14 days after antigen administration. As discussed in Example 11, Balb / cByJ mice were vaccinated with liposomes containing bFGF (bFGF liposomes) or without liposomes (control liposomes) according to the protocol outlined in the Methods section. Inoculate or PBS (PBS)
Treated. On day 35, 1 × 10 ⁶ LLC-LMs were challenged subcutaneously on the back of the mice. The data shown are the mean tumor volume ± 1 S.D. of the mean measured 14 days post-challenge for each of the three vaccination groups. D. It is.

FIGS. 16 (a) to (c) are graphs showing the immunoreactivity of a test peptide to an autologous peptide fragment of VEGF. As discussed in Example 13, Balb / cByJ mice were vaccinated with liposomes containing VEGF peptides 1-3 incorporated into liposomes by conjugation according to the protocol outlined in the present invention. Sera from individual mice were collected on day 35, serially diluted, and analyzed for reactivity to autologous peptide fragments of VEGF by measuring absorbance in an ELISA assay. The NMS control shows the reactivity of sera obtained from non-vaccinated mice, and does not differ from the reactivity of sera obtained from mice vaccinated with the liposome control.

FIGS. 17 (a) and 17 (b) are graphs showing the immunoreactivity of a test peptide against an autologous peptide fragment of VEGF. As discussed in Example 13, Balb / cByJ mice were vaccinated with liposomes containing VEGF peptides 2 and 3 incorporated into liposomes by simple encapsulation according to the protocol outlined in the present invention. Sera from individual mice were collected on day 35, serially diluted, and the V was determined by measuring the absorbance in an ELISA assay.
EGF was analyzed for reactivity to autologous peptide fragments. NM
The S control shows the reactivity of sera obtained from non-vaccinated mice and is not different from the reactivity of sera obtained from mice vaccinated with the liposome control.

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

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

FIG. 20 is a graph showing the results of an experiment performed to demonstrate the induction of an antibody response to an FGF-2 heparin binding domain peptide. Mice were treated with FGF-2 peptide covalently complexed to liposome vesicles every 2 weeks for 6 weeks. Serum from mice treated with the heparin binding domain peptide was FG by ELISA.
Reacted with F-2 heparin binding domain peptide (◇) and native FGF-2 molecule (□). This serum contains the receptor binding domain peptide (O) or other VEGF (
No immunoreactivity was shown for Δ).

FIGS. 21a-c show inhibition of B16BL6 tumor growth and angiogenesis in liver sponge grafts. Gelatin sponges containing 5 × 10 ⁴ B16BL6 tumor cells were implanted into the liver of mice pre-treated with L (HBD), L (RBD) or L (LA). Fourteen days after implantation, sponges were removed and sectioned for histology. H & E staining of sponges removed from mice treated with L (LA) (a) or L (RBD) (b) revealed B16BL6 tumor growth and angiogenesis, while treated with L (HBD) (c) Sponge removed from mice contained a large number of red blood cells lacking characteristic peripheral structures.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 7/06 A61P 7/06 9/00 9/00 15/00 15/00 17/00 17/00 17 / 06 17/06 19/00 19/00 19/02 19/02 27/02 27/02 27/06 27/06 35/00 35/00 35/02 35/02 35/04 35/04 // C07K 14/475 ZNA C07K 14/475 ZNA 14/50 14/50 17/02 17/02 (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ) MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT , LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Madsen, John United States of America Maryland 21758 Knoxville Petersville Road 3738 F-term (reference) 4C085 AA03 AA04 BB07 BB14 BB15 BB24 DD62 EE01 EE06 FF02 FF03 FF13 FF14 FF20 4H 045 AA11 AA20 AA30 BA10 BA61 CA40 DA01 EA28 FA72 FA74

Claims (46)

[Claims] 1. An immunogenic composition comprising an immunogenic peptide fragment of fibroblast growth factor and a pharmaceutically acceptable carrier. 2. The composition according to claim 1, wherein said immunogenic peptide corresponds to the heparin binding domain of fibroblast growth factor. 3. The composition according to claim 1, wherein the amino acid sequence of the immunogenic peptide comprises the sequences of SEQ ID NOs: 1 and 2. 4. The composition of claim 1, wherein said pharmaceutically acceptable carrier comprises a liposome, colloidal gold and a carrier protein. 5. The method according to claim 1, wherein the carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, thyroglobulin, serum albumin, γ-globulin, syngeneic cells, and D.
The composition according to claim 4, comprising a polymer of-and / or L-amino acids. 6. The composition according to claim 4, further comprising an adjuvant, a preservative, a diluent, an emulsifier and a stabilizer. 7. The adjuvant wherein a lipid-soluble 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 mannan, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin derivative adjuvant, dead pertussis, lipopolysaccharide of gram-negative bacteria, polymer anion, dextran sulfate, inorganic gel, alum, aluminum hydroxide and phosphorus 7. The composition according to claim 6, wherein the composition is selected from the group consisting of aluminum oxides. 8. The composition of claim 1, further comprising a hydrophobic moiety associated with said immunogenic peptide. 9. The composition of claim 8, wherein said hydrophobic moiety comprises at least one long chain fatty acid having at least 10 carbon atoms in the lipid backbone. 10. The composition of claim 8, wherein said hydrophobic moiety is selected from the group consisting of palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. 11. An immunogenic composition comprising an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier. 12. The composition according to claim 11, wherein said immunogenic peptide fragment corresponds to the receptor binding domain of vascular endothelial growth factor. 13. The composition according to claim 11, wherein the amino acid sequence of the immunogenic peptide comprises the sequence of SEQ ID NOs: 3-9. 14. The composition according to claim 11, wherein said pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier protein. 15. The carrier protein according to claim 12, wherein said carrier protein is maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, thyroglobulin, serum albumin, γ-globulin, syngeneic cells, and D- and / or L-. 15. The composition of claim 14, comprising a polymer of an amino acid. 16. The composition according to claim 14, further comprising an adjuvant, a preservative, a diluent, an emulsifier and a stabilizer. 17. The adjuvant is a fat-soluble muramyl dipeptide derivative, a nonionic block polymer, aluminum hydroxide, aluminum phosphate, and lipid A.
Incomplete Freund's adjuvant, complete Freund's adjuvant, polydisperse β- (
1,4) bound acetylated mannan, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin derivative adjuvant, dead pertussis, lipopolysaccharide of gram-negative bacteria, polymer anion, dextran sulfate, inorganic gel, alum, aluminum hydroxide and 17. The composition according to claim 16, wherein the composition is selected from the group consisting of aluminum phosphate. 18. The composition of claim 14, further comprising a hydrophobic moiety associated with said immunogenic peptide. 19. The composition of claim 18, wherein said hydrophobic moiety comprises at least one long chain fatty acid having at least 10 carbon atoms in the lipid backbone. 20. The composition of claim 18, wherein said hydrophobic moiety is selected from the group consisting of palmitic acid, stearic acid, myristic acid, lauric acid, oleic acid, linoleic acid, and linolenic acid. 21. An immunogenic composition comprising an immunogenic peptide fragment of fibroblast growth factor and vascular endothelial growth factor and a pharmaceutically acceptable carrier. 22. The immunogenic peptide fragment of fibroblast growth factor corresponds to the heparin-binding domain of fibroblast growth factor and the immunogenic peptide fragment of vascular endothelial growth factor binds to receptor of vascular endothelial growth factor. 22. The composition according to claim 21 corresponding to a domain. 23. The composition of claim 22, wherein said pharmaceutically acceptable carrier comprises a liposome, colloidal gold and a carrier protein. 24. The carrier protein comprising maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, thyroglobulin, serum albumin, γ-globulin, syngeneic cells, and D- and / or L-. 24. The composition of claim 23 comprising a polymer of an amino acid. 25. The composition according to claim 23, further comprising an adjuvant, a preservative, a diluent, an emulsifier and a stabilizer. 26. The adjuvant wherein a lipid-soluble 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 mannan, polyoxyethylene-polyoxypropylene copolymer adjuvant, saponin derivative adjuvant, dead pertussis, lipopolysaccharide of gram-negative bacteria, polymer anion, dextran sulfate, inorganic gel, alum, aluminum hydroxide and 26. The composition according to claim 25, wherein the composition is selected from the group consisting of aluminum phosphate. 27. A human or animal cancer or hyperproliferative comprising administering to a human or animal an effective amount of a composition comprising an immunogenic peptide fragment of fibroblast growth factor and a pharmaceutically acceptable carrier. How to treat a disease. 28. The method of claim 27, wherein said immunogenic peptide fragment corresponds to the heparin binding domain of fibroblast growth factor. 29. The method of claim 27, wherein the amino acid sequence of said immunogenic peptide fragment comprises the sequences of SEQ ID NOs: 1 and 2. 30. The method of claim 27, wherein said pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier protein. 31. The carrier protein comprising maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, thyroglobulin, serum albumin, γ-globulin, syngeneic cells, and D- and / or L-. 28. The method of claim 27, comprising a polymer of amino acids. 32. The method of claim 27, further comprising an adjuvant, a preservative, a diluent, an emulsifier, and a stabilizer. 33. The hyperproliferative disease wherein the hemangiomas, solid tumors, blood-derived tumors,
Leukemia, metastasis, telangiectasia, psoriasis, scleroderma, purulent granulomas, myocardial angiogenesis,
Crohn's disease, plaque angiogenesis, cerebral arteriovenous malformation, corneal disease, rubeosis, angiogenic glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, 3. Including helicobacter-related diseases, fractures, keloids, angiogenesis, hematopoiesis, ovulation, menstruation, placental formation and cat scratch fever.
7. The method according to 7. 34. A human or animal cancer or hyperproliferative disease 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. How to treat. 35. The method of claim 34, wherein said immunogenic peptide fragment corresponds to the receptor binding domain of vascular endothelial growth factor. 36. The method of claim 34, wherein the amino acid sequence of said immunogenic peptide fragment comprises the sequence of SEQ ID NOs: 3-9. 37. The method of claim 34, wherein said pharmaceutically acceptable carrier comprises a liposome, colloidal gold and a carrier protein. 38. The carrier protein may be maltose binding protein, bovine serum albumin, keyhole limpet hemocyanin, ovalbumin, flagellin, thyroglobulin, serum albumin, γ-globulin, syngeneic cells, and D- and / or L-. 35. The method of claim 34, comprising a polymer of amino acids. 39. The method of claim 34, further comprising an adjuvant, a preservative, a diluent, an emulsifier, and a stabilizer. 40. The hyperproliferative disease wherein the hemangiomas, solid tumors, blood-derived tumors,
Leukemia, metastasis, telangiectasia, psoriasis, scleroderma, purulent granulomas, myocardial angiogenesis,
Crohn's disease, plaque angiogenesis, cerebral arteriovenous malformation, corneal disease, rubeosis, angiogenic glaucoma, diabetic retinopathy, retrolental fibroplasia, arthritis, diabetic neovascularization, macular degeneration, wound healing, peptic ulcer, 4. Helicobacter-related diseases, including fractures, keloids, angiogenesis, hematopoiesis, ovulation, menstruation, placentation, and feline scratch fever.
4. The method according to 4. 41. A method of treating a human or animal in need of an immune response to a growth factor, comprising administering to the human or animal an effective amount of a growth factor composition, wherein the composition comprises a human or animal. Or a therapeutic method comprising an immunogenic peptide fragment of fibroblast growth factor and a pharmaceutically acceptable carrier such that when administered to an animal, the composition is immunogenic for fibroblast growth factor. 42. The method of claim 41, wherein said immunogenic peptide comprises the sequences of SEQ ID NOs: 1 and 2. 43. The method of claim 41, wherein said pharmaceutically acceptable carrier comprises liposomes, colloidal gold and carrier protein. 44. A method of treating a human or animal in need of an immune response to a growth factor, comprising administering to the human or animal an effective amount of a growth factor composition, wherein the composition comprises a human or animal. A method of treatment comprising an immunogenic peptide fragment of vascular endothelial growth factor and a pharmaceutically acceptable carrier such that when administered to a vascular endothelial growth factor, the composition is immunogenic with respect to vascular endothelial growth factor. 45. The method of claim 44, wherein said immunogenic peptide comprises the sequence of SEQ ID NOs: 3-9. 46. The method of claim 44, wherein said pharmaceutically acceptable carrier comprises a liposome, colloidal gold and a carrier protein.