Treatment and/or prevention of non-infectious medical conditions using antibody-containing compositions

Field of invention The present invention relates to the treatment and / or prevention of non-infectious medical conditions, preferably by topical application, using antibody-containing compositions. In a particular embodiment, the invention comprises the step of administering an antibody-containing milk product and / or egg product, preferably by topical application, for the treatment and / or prevention of cancer or non-cancerous or hyperproliferative disorders. Regarding the method.

Background of the invention Treatment of many serious medical conditions is almost universally performed systemically. However, there are certain medical conditions for which non-systemic (eg, topical) treatment may be more appropriate, such as, but not limited to, the skin, gastrointestinal (GI) tract (eg, oropharynx), eyes, ears. , Such as those affecting the nasopharynx, bladder, vagina and neck.

Skin cancer accounts for almost 81% of all new cancer diagnoses in Australia each year. There are three main types of skin cancer: basal cell carcinoma and squamous cell carcinoma (also known as nonmelanoma skin cancer) and melanoma. Nonmelanoma skin cancer is Australia's most common cancer (more than 374,000 new cases each year), and although it is the least life-threatening, some cases can be fatal.

GI cancers include a wide variety of cancers, including colorectal cancer. Globally, colorectal cancer (colorectal cancer) is the third most common cancer in men and women, accounting for 13% of all cancers. In Australia, this cancer is the second most common cancer in both men and women. Approximately 20% of patients have a metastatic disease (spread from the large intestine) at diagnosis, and 50% of patients with total colorectal cancer develop metastases and eventually die of this disease. Approximately 56,000 people die from colorectal cancer each year in the United States, with a mortality rate of over 100,000 patients annually in Europe.

Colorectal cancer is a solid tumor (proliferation) that first develops in the innermost layer of the intestine (ie, the inner lining of the endothelium) and can grow in part or all of the other layers. Colon cancer begins in a benign neoplasm and progresses to adenocarcinoma through a stepwise histological progression sequence, progressing from either an adenoma or a hyperplastic polyp / serrated adenoma.

Colorectal cancer usually begins with the growth of polyps. In 1994, more than 2,000,000 colonoscopies were performed in the United States, of which more than 650,000 patients underwent polypectomy. Early detection and removal of polyps significantly reduces the incidence of colorectal cancer. However, about 50 percent of people aged 60 years have at least one adenomatous (potentially cancerous) polyp with a diameter of 1 cm or more.

Most patients with colorectal cancer have treatment that can assist in the prevention and / or treatment of colorectal cancer without the need for surgery because it indicates a late stage of the disease, or after surgery, eg, polypectomy. It would be desirable to reduce the risk of recurrence after (polpectomy).

Esophageal cancer is currently the ninth most common cancer in adults in the United Kingdom. The precursor to esophageal cancer is Barrett's esophagus. In Barrett's esophagus, the cells in the inner layer of the esophagus change to resemble the cells in the inner layer of the stomach. It can develop after prolonged gastric reflux. Has Barrett's esophagus People are 125 times more likely to develop esophageal cancer than the average person.

Head and neck cancers, such as oral and pharyngeal cancers, are the sixth most common malignancies reported worldwide and have very high mortality rates for all types of malignancies. .. Approximately 90 percent of head and neck cancers are altered squamous epithelial cells. Chemotherapy and surgical procedures have improved considerably, but the disease is often particularly treated, as most patients with advanced disease have secondary tumors and also suffer from other comorbidities. Is difficult.

Bladder cancer is the fourth most common cancer in men, with more than 10,000 people diagnosed with the disease each year in the United Kingdom. Bladder cancer is rare in people under the age of 40. The average age at diagnosis is 65 years. By the time of diagnosis, about 85% of bladder cancers still remain localized to the bladder. Bladder cancer usually begins first in the mucosa and grows only towards the inside of the bladder for some time.

Cancer of the eye is uncommon, but is a series of serious conditions that require effective treatment and prevention. Cancer can occur in any part of the eye. The most common malignant primary intraocular tumor in adults is melanoma. Most attention is paid to posterior choroid plexus melanoma, which can also occur in the iris and ciliary body. The most common eyelid cancer is basal cell carcinoma. The cancer can grow around the eyes, but rarely spreads to other parts of the body. Other common types of eyelid carcinoma include squamous cell carcinoma, sebaceous carcinoma and malignant melanoma.

Uterine cancer is the most common gynecologic cancer. Most cancers of the uterus are cancers of the lining of the uterus (endometrium). Cancer can also occur in the muscle layer of the uterus. Other less common types of uterine cancer are adenosquamous carcinoma, papillary serous cancer, and rarely clear cell carcinoma or uterine sarcoma. These cancers are invasive and prone to metastasis. Cervical cancer is less common than uterine cancer, but typically occurs first in the squamous epithelial cells that line the outer surface of the cervix.

It may be desirable to have treatment that can assist in the prevention and / or treatment of the above and related conditions without the need for surgery, or to reduce the risk of recurrence after surgery.

References such as literature, act, materials, devices, treatises, etc. are included herein solely for the purpose of providing background to the present invention. Some or all of these, which existed prior to the priority date of each claim of the present application, constitute the basis of prior art in the art related to the present invention, or are common knowledge. That is neither suggested nor presented.

<p> The present invention is based on antibody-containing milk or egg products suitable for use in the treatment of non-infectious medical conditions, preferably for use by topical application.</p><p> In a first aspect, the invention provides the use of antibody-containing milk products or egg products for the manufacture of reagents for the prevention and / or treatment of non-communicable medical conditions. This aspect of the invention is also a method of preparing a pharmaceutical in the form of a food, specialty nutritional food or pharmaceutical for the prevention and / or treatment of non-infectious medical conditions, wherein the antibody-containing milk product or egg product is. Provided are methods that include vaccination of mammals or birds to be produced. In one embodiment of the invention, the mammal is selected from the group consisting of cows, sheep, pigs and goats. In another form, birds are selected from the herd consisting of chickens, geese and ducks. In another embodiment of the invention, the step of vaccination involves administering an immunogenic protein or peptide to a mammal or bird and said the immunogenic protein or peptide. Tide can elicit an immune response against molecules of mammalian origin, such as, but not limited to, molecules found on the surface of mammalian cells (such as, but not limited to, cancer cells).</p><p> In a second aspect, the invention provides a method of preventing and / or treating a patient's non-communicable medical condition, comprising administering to the patient an antibody-containing milk product and / or egg product. In one embodiment of the invention, the milk product and / or egg product refers to a molecule of mammalian origin, such as, but not limited to, a molecule found on the surface of a mammalian cell (such as, but not limited to, a cancer cell). Contains antibodies.</p><p> In a third aspect, the invention comprises an antibody-containing milk product or product for the prevention and / or treatment of a non-infectious condition, comprising the step of vaccination of a mammal or bird with an immunogenic protein or peptide. A method for preparing an egg product is provided.</p><p> The present invention provides antibodies, nutritional compositions and pharmaceutical compositions and kits comprising antibody-containing milk products and / or egg products according to the present application and / or antibodies produced according to the methods according to the present application.</p><p> The present invention also provides the use of anti-EphB4 antibodies for use in the treatment and / or prevention of esophageal conditions, and for use in the diagnosis and monitoring of such conditions. In certain embodiments of the invention, methods of diagnosing esophageal status include examining EphB4 expression in esophageal samples, where detection of increased EphB4 expression compared to normal esophageal samples indicates the presence of esophageal status. Shown. In certain embodiments, the condition of the esophagus is selected from the group consisting of Barrett's esophagus and esophageal cancer. The present invention also provides a kit for performing this method.</p><p> The present invention also provides the treatment and / or prevention of colorectal polyps and the use of anti-EphB4 antibodies in postoperative treatment after polypectomy in colorectal cancer.</p>

<figref num="1">FIG. 1 shows the relative mRNA levels of EphA1 (A), EphA2 (B) and EfnA1 (C) in various tissue groups. The box indicates the range from the 25th to the 75th percentile (interquartile). The median value is indicated by a horizontal bar in each box. The whiskers indicate the outer levels of the 25th to 75th percentiles. asterisk(^*) Indicates that p &lt;0.017 for normal.</figref><figref num="2">FIG. 2 shows the relative mRNA levels of EphB1 (A), EphB2 (B), EphB4 (C) and EfnB2 (D) in various tissue groups. The box indicates the range from the 25th to the 75th percentile (interquartile). The median value is indicated by a horizontal bar in each box. The whiskers indicate the outer levels of the 25th to 75th percentiles. asterisk(^*) Indicates that p &lt;0.017 for normal.</figref><figref num="3">FIG. 3 shows standardized EphB4 mRNA levels in Het1A (esophageal epithelial cells) and OE33 (esophageal cancer) cell lines.</figref><figref num="4">FIG. 4 shows the sequence of human EphB4 protein (SEQ ID NO: 1).</figref><figref num="5">FIG. 5 shows the sequence of mouse EphB4 protein (SEQ ID NO: 2).</figref><figref num="6">FIG. 6 shows the sequence of bovine EphB4 protein (SEQ ID NO: 3).</figref>

Detailed description of the invention The present invention seeks to provide a method of treating and / or preventing a non-infectious condition by non-systemic administration of a biologically active agent. For example, applying an anticancer drug directly to cancer may be more convenient than applying systemic treatment. GI status and, in the case of cancer, the GI lumen Administration of a biologically active agent via is may be more convenient than performing systemic treatment. In GI cancer, for example, a biologically active molecule should specifically target any abnormal cell in the inner layer of the GI tube and suppress its growth.

It is generally accepted that all types of cancer treatment have some side effects. Systemically administered monoclonal antibodies are more developed than other treatments because they are derived from proteins that are naturally present in the body and should target specific cells and not attack non-target cells. It is believed to have few side effects. However, systemic administration of the antibody is often accompanied by side effects such as fever, chills and tremors, nausea, itchy rash, headache; and in some cases wheezing and decreased blood pressure.

Non-systemic treatment of the disease (eg, topical treatment) is to directly target diseased cells, reduce side effects, and substantially suppress drug therapy that has a detrimental effect on non-target tissue. desirable. Not all medical conditions are sensitive to topical treatment, but certain non-infectious medical conditions that affect the skin, GI ducts, eyes, ears, nasopharynx, bladder, vagina and neck are particularly treated in this manner. Suitable for.

Substances in the GI canal and nasopharyngeal cavity remain effective outside the body, and the integrity of the GI canal and nasopharynx prevents the organism from escaping into the rest of the organism. This integrity is provided by the tight junctions found between the cells in the inner layers of these parts of the body. The present invention utilizes this separation and similar barriers found in the bladder, eyes, vagina, uterus and neck to enable treatment of specific targets without invading other parts of the body. is there. For example, when treating a condition with a biologically active drug in a systemic manner, it is necessary to balance the effective dose with a dose that has a significant effect on non-target sites throughout the body. This limitation is greatly reduced by the method of the present invention.

The skin likewise provides a barrier between the body and the outside. Therefore, direct application of a biologically active drug to a skin condition provides similar advantages over systemic administration of the drug.

Antibodies, in particular, are large molecules that normally cannot pass through the GI ducts, skin, lining of the bladder, cornea or mucous membranes (such as those in the lining of the vagina and neck) on their own. Therefore, administration of the antibody to a patient is usually done parenterally, eg, subcutaneously, intramuscularly or intravenously, without limitation. Such a mode of administration allows the molecule to enter the body as is and be delivered throughout the body in a systemic manner. While some small molecules can be delivered systemically by oral administration, the present invention relates to antibodies and related molecules that require methods such as injection that functionally enter the body as is.

Currently, several research groups and companies have been working on the prevention of various GI tube infections caused by infectious organisms such as Clostridium difficile, Cryptosporidium, Escherichia coli, rotavirus, H. pylori, Taiwan E71 virus and charcoal. Milk products for administration have been developed by hyperimmunizing cattle with a vaccine to produce antibodies against these infections. However, all such products are intended for use in the prevention or treatment of infectious diseases. The present invention looks ahead beyond the application of infectious disease control and is a solution to problems that have not yet been addressed in the art, i.e., non-communicable medical conditions such as, but not limited to, cancer, precancerous. It provides a simple and effective prophylactic and / or treatment approach for the non-systemic treatment of conditions and non-cancerous conditions (such as hyperproliferative disorders). Hyperproliferative or precancerous conditions affecting epithelial cells, such as psoriasis, vitiligo, atopic dermatitis, or hyperproliferative or UV-responsive skin disorders, are also suitable for prevention and / or treatment by the methods of the invention. Candidate.

The present invention provides antibody-containing milk and / or egg products that can be used to treat and / or prevent non-infectious conditions in the skin, GI tubes, eyes, ears, nasopharynx, bladder, vagina, uterus or neck. provide.

Colorectal cancer generally first occurs as a polyp, which is reachable from the GI lumen and provides an opportunity for a non-systemic approach to the treatment of the disease. Also, some other cancerous and non-cancerous conditions of the GI canal may be sensitive to this approach. An example of a non-cancerous state of GI in which the present invention may be useful is Barrett's esophagus (discussed below).

As used herein, the term "antibody" refers to any form and fragment of an antibody such as, but not limited to, mammalian antibody, avian antibody, monoclonal antibody, polyclonal antibody, multispecific antibody, chimeric antibody, single chain. It shall include antibodies, humanized antibodies and fragments thereof. For example, antibody fragments include, but are not limited to, Fv, Fab, Fab', F (ab').₂, And CDRs (Complementarity determining Regions). Also included are antibodies of all immune classes, such as, but not limited to, IgA, IgE, IgG, IgM and IgY. In addition, degradation products and fragments of antibodies that maintain their ability to bind antigen in a specific manner are included. Degradation can occur, for example, when the antibody is passing through the GI tube.

As used herein, the term "molecules on the surface of a mammalian cell" presents at least a protein normally present on the extracellular surface of a mammalian cell, or at least a portion on the extracellular surface of a mammalian cell. It shall include the protein (eg, transmembrane protein).

Such proteins may include post-translational modifications capable of eliciting an immune response. Typical post-translational modifications include, but are not limited to, glycosylation and phosphorylation. The molecule itself may not be immunogenic due to its small size, but methods such as conjugation to carrier proteins can be used to increase the immunogenicity of the mammalian molecule in question. it can. The molecule can also be a mutant form of a protein normally present on the extracellular surface.

As used herein, the term "GI tube" shall include the nasopharynx, oral cavity, esophagus, stomach, duodenum, small intestine, colon (colon), rectum and anus.

As used herein, the term "non-communicable pathology" is intended to include, but is not limited to, conditions such as cancer, precancerous conditions, non-cancerous hyperproliferative diseases and overgrowth of "autologous" cells. To do. In a particular embodiment of the invention, the cancer can be GI cancer or skin cancer. Cellular overgrowth can include conditions such as, but not limited to, Barrett's esophagus, psoriasis, vitiligo, atopic dermatitis, and hyperproliferative or UV-responsive skin disorders. The term includes both inflammatory and non-inflammatory conditions. Many inflammatory diseases will be known to those of skill in the art. Such disorders include, but are not limited to: migraine, rheumatoid arthritis, asthma, inflammatory bowel disease, agitation, induced vomiting, pain, headache, migraine, multiple sclerosis, cardiovascular changes: , Edema, asthma / bronchial hypersensitivity and other respiratory diseases such as allergic rhinitis, intestinal inflammatory diseases (eg ulcerative colitis and Crohn's disease), eye damage and eye inflammatory diseases Be done. Non-inflammatory conditions include, but are not limited to: fibromyalgia and non-inflammatory neuropathy.

As used herein, the term "non-infectious" shall include conditions that are not caused by the invasion or transmission of a foreign entity. Draw that state in a non-infectious state The awakening entity is generally an integrated entity that cannot invade other cells.

As used herein, the term "GI cancer" is intended to include any cancer that develops and infringes in the GI duct. Such cancers include, but are not limited to, anal cancer, bile duct cancer, colon-rectal (colon and / or rectal) cancer, esophageal cancer, gallbladder cancer, gastric cancer, liver cancer (hepatoma), pancreatic cancer and small intestinal cancer. Cancers of the oropharyngeal, mouth, tongue and nose include. Of these, the most suitable cancers for treatment by the method of the present invention are those that can be reached directly from the GI lumen or those that invade the GI lumen. It also includes cancers that originate in the viscera or other organs that can be treated locally and metastasize into the GI duct.

As used herein, the terms "non-cancerous" and "pre-cancerous" are intended to include non-infectious medical conditions for which treatment may be desired but not cancer. For example, a condition such as Barrett's esophagus is a condition in which abnormal cells are generated on the medial lining of the lower part of the esophagus, probably due to the regurgitation of gastric contents into that region of the esophagus. Barrett's esophagus is not a cancerous condition, but over time it can lead to the development of cancer in the lower esophagus in some cases. Therefore, Barrett's esophagus can also be considered a precancerous condition, and its treatment is therefore desirable. The present invention reports for the first time the upregulation of EphB4 in Barrett's esophagus, which provides a model showing certain aspects of the invention.

As used herein, the term "skin cancer" is intended to include any cancer that develops on the skin. Such cancers include, but are not limited to, basal cell carcinomas, squamous cell carcinomas (also known as nonmelanoma skin cancers) and melanomas.

As used herein, the term "patient" shall include humans and animals, in particular those who have or are at risk of developing the condition in the GI, skin, eyes, bladder or neck. .. In most embodiments of the invention, the patient is a mammal, but usually a human.

As used herein, the term "administration" shall be limited to non-systemic administration, eg, but not limited to topical application, oral administration and oral ingestion. Also, the form of introducing the antibody-containing milk product or egg product directly into the patient's GI tube, nose, bladder, eyes or vagina / neck, eg, as a liquid in the esophagus or in the form of a suppository in the colon. Incorporation is included, or as an enema, or directly into the bladder via a catheter. Also, pure immunoglobulin preparations can be prepared from antibody-containing milk products or egg products, which can be applied directly onto tumor cells, for example, in the form of "colon perfusion" in the colon, and in the eye. It can be used in the form of "eye irrigation" or for other suitable procedures. There are many potential formulations for skin application.

The term "non-systemic administration" shall exclude systemic routes of administration (eg, intravenous administration, etc.). Other routes of administration are well known to those of skill in the art.

The amount of antibody required for administration is described as "therapeutically effective amount". A "therapeutically effective amount" is an amount that elicits a biological or medical response to a tissue or system of interest. For example, in the case of treatment of GI cancer, this is the amount of antibody (contained in the nutritional or pharmaceutical matrix) that can slow or suppress the growth of GI cancer cells. In addition, the "therapeutic effective amount" can reduce the number of existing GI cancer cells.

In certain embodiments of the invention, the amount of antibody administered to a patient can be on the order of 0.1-30 g (dry weight) antibody / day. For certain applications, the antibody dose is 0. 05-2.0 g (dry weight) antibody / kilogram body weight / day can be calculated using the formula. Using this formula, infants can be administered 0.2-5 g (dry weight) antibody / day and adults can be administered 0.5-15 g (dry weight) antibody / day.

As used herein, the term "milk product" is intended to include any form of milk of any mammalian species. Such forms include, but are not limited to, first milk, first milk-derived products, milk and milk-derived products. In addition, hyperimmune milk and products derived from hyperimmune milk are also included in the term "milk products". It is generally assumed that cows will be the source of milk products. However, additional species such as, but not limited to, sheep, pigs and goats can be used.

The term "first milk", as used herein, is first milk milk; processed first milk milk (first processed to partially or completely remove one or more of fat, cell debris, lactose and casein). Milky milk, etc.); as well as, for example, primary milk or processed primary milk dried by freeze-drying, spray-drying or other drying methods known in the art. First milk is generally collected from mammals such as cows within 5 days of delivery.

As used herein, the term "egg product" shall include all forms of eggs of any avian species. Such forms include, but are not limited to, whole egg, yolk, egg or yolk-derived products. Although chickens are generally assumed to be the source of egg products, additional or alternative species such as geese and ducks may also be used.

Other products incorporating the antibodies that are included in the present invention include, but are not limited to, fractions and / or concentrated immunoglobulin fractions; pure antibody fractions; specific antibody fractions (eg, affinity). Isolated by chromatography); long-lived liquids, spray-dried or lyophilized powders; fresh dairy products; nutritional foods with high viscosity to prolong contact time in the oropharynx and esophagus (eg, "Sippy" Food (sip feeds) "). Also, the antibodies of the invention are reagents that allow the antibody to be delivered in active form to various prophylactic / treatment sites, such as, but not limited to, the lower GI canal, eyes, bladder, uterus, cervix and skin. It is envisioned that it can be incorporated into compositions and products.

In a first aspect, the invention provides the use of antibody-containing milk products or egg products for the manufacture of reagents for the prevention and / or treatment of non-communicable medical conditions. This aspect of the invention is also a method of preparing a food product of pharmaceutical or medical benefit for non-systemic administration for the prevention and / or treatment of non-infectious medical conditions, the antibody-containing milk product. And or a method comprising the step of vaccination of an immunogenic protein or peptide to a mammal or bird so that an egg product is produced.

Vaccinated mammals may be capable of producing commercially feasible amounts of milk. Therefore, mammals are expected to be selected from the herd consisting of cattle, sheep, pigs and goats. Vaccinated birds can be capable of laying commercially viable amounts of eggs and are therefore usually chickens, geese or ducks.

The step of vaccination involves administering an immunogenic protein or peptide to a mammal or bird, and the immunogenic protein or peptide can elicit an immune response against a mammalian-derived molecule. In one embodiment of the invention, mammalian-derived molecules can be found on the surface of mammalian cells (such as cancer cells). In other forms of the invention, mammalian-derived molecules are mutant proteins or proteins not normally found on the surface of mammalian cells. Can be.

For successful treatment with antibody-containing milk or egg products, the milk or egg product ideally contains an antibody (immunoglobulin) that has a high affinity for the target molecule. Ideally, therefore, in a vaccinated mammal or bird, it is ideal to provide an antigen that can elicit such a humoral response without causing the condition in the mammal, its offspring or birds.

Vaccination of mammals or birds ideally involves administration of an antigen that results in the production of highly compatible and selective antibodies to the target molecule. A suitable antigen can be obtained by several procedures. In one method, a pure ligand is used, the ligand is covalently attached to a suitable chromatography matrix, which can be used to affinity purify the target molecule from a crude cell extract to purify the target molecule. Alternatively, the target molecule can be purified using immunoaffinity purification methods with specific polyclonal antibodies covalently attached to a suitable chromatography matrix. In yet another method, the target molecule can be made by recombinant techniques according to standard procedures in the art and then purified.

It is envisioned that antibodies directed to molecules on the surface of cancer cells may provide effective therapies for preventing and / or treating the cancer from which the cells were harvested.

Identification of cancer-specific cell surface molecules is the goal of a research group attempting to create a list of cell surface proteomes for various cancer cells. One approach for identifying such molecules is to biotinify the cell surface and then capture the biotinylated protein with avidin. The captured protein is then subjected to identification techniques such as two-dimensional polyacrylamide gel electrophoresis and / or partial sequencing.

In addition, such studies make it possible to identify potential targets for the methods of the invention. Molecules that exhibit a limited expression pattern can be particularly important, especially if they are more expressed on a population of target cells (such as cancer cells). Alternatively, molecules that play a role in tumorigenesis are also potential targets.

One of the potential target molecules is vascular endothelial growth factor (VEGF), which is not a cell surface molecule but has a role in tumor angiogenesis. The onset of angiogenesis is considered an early event of tumorigenesis and can facilitate tumor progression and metastasis. Several growth factors with angiogenic activity have been reported. Such include fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and VEGF. VEGF is a dimeric glycoprotein that has structural homology with PDGF. Several variants of VEGF produced by alternative splicing have been reported. It has been speculated that VEGF may function as a tumor angiogenesis factor in vivo because the expression pattern of VEGF is consistent with its role in embryonic angiogenesis. VEGF mRNA is formed in some primary tumors, VEGF is produced by tumor cell lines in vitro, and VEGF mitogen activity appears to be confined to endothelial cells. In addition, monoclonal anti-VEGF antibodies have been shown to reduce tumor angiogenesis.

Additional antigens that may provide useful targets for the methods of the invention include molecules found on the surface of hyperproliferating cells. For example, in psoriasis, T cells of the immune system recognize proteins / antigens in the skin and attack the areas where the proteins are found, causing too rapid proliferation of new skin cells and the promotion of painful scaly lesions. It is considered to be an autoimmune disease. This lesion is characterized by overgrowth of keratinocytes and accumulation of activated T cells in the epidermis of psoriatic lesions. According to the skin protein / antigen to which T cells are directed, the present invention Target molecules useful in the law may be provided. For example, MUC18 (also known as Mel-CAM or CD146) shows some association with psoriasis and some cancers, thus providing a potential target. Similarly, other hyperproliferative disorders also have specific antigens that can be used in the methods of the invention.

A large group of latent target molecules is the Eph receptor protein family. The Eph receptor is composed of the largest subgroup of the receptor tyrosine kinase (RTK) family. RTKs are transmembrane proteins that have an extracellular domain that can recognize signals from the cell's environment and proliferate cells by acting directly on gene transcription or indirectly on the production of second messengers. And can affect survival.

We have identified EphB4 as an example of a potential target for the treatment of a range of locally treatable cancers, such as some GI, nasopharyngeal, skin, uterine and bladder cancers (see Examples). .. In addition, EphB1, EphB2, EfnA1 and EfnB2 were identified in the Eph / ephrin group as potential target proteins. Although the discussion is focused on EphB4 below, the methodology described is equally applicable to other members of the Eph / ephrin family as well as other cell surface molecules.

EphB4 binds to ephrin-B1 and B2 and may play a role in events that mediate differentiation and development. It is highly expressed in the placenta and series of primary tissues and malignant cell lines. EphB4 is expressed in the fetal brain (not expressed in the adult brain), as well as in primitive and myeloid hematopoietic cells (not expressed in the lymphatic system). This includes several carcinomas: breast cancer (mRNA detected in MCF7, MDA-MB-231, SKBR, T47D, BT20 and BT474 human cell lines), liver cancer, gastrointestinal cancer, leukemia, prostate, lung (3x down) (Regulatory), and present in carcinoma. One study of EphB4 expression in colon tumors found that expression was upregulated&gt; 1.5-fold in 82% (51/62) of samples and&gt; 2-fold in 63% (39/62) of samples. It was. It was also overexpressed in ovarian cancer and upregulated in glial blastoma (&gt; 5-fold). However, other studies have shown that EphB4 expression is down-regulated 2.2-fold in kidney cancer. In addition, low EphB4 expression in some colorectal cancers is associated with short survival of patients, and it has been reported that EphB4 expression varies in breast cancer. Therefore, there is no consistent association between EphB4 expression and cancer progression in surviving patients.

The results shown in the Examples below are the first to show that EphB4 expression is increased in Barrett's esophagus and associated esophageal adenocarcinoma compared to expression levels in normal esophageal tissue. Therefore, antibodies directed to EphB4 may find application in the prevention and / or treatment of esophageal conditions such as Barrett's esophagus and esophageal adenocarcinoma. In addition, it is predicted that EphB4 may provide markers for the development and / or progression of Barrett's esophageal and / or esophageal adenocarcinoma. Accordingly, the present invention provides for the treatment and / or prevention of esophageal conditions and the use of anti-EphB4 antibodies in the diagnosis of such conditions. It is also envisioned that patients may be given anti-EphB4 antibodies to prevent or delay the development of conditions such as Barrett's esophagus and / or esophageal adenocarcinoma after increased EphB4 expression is detected in the patient's esophagus. To.

Therefore, the present invention comprises providing a sample of patient-derived esophageal cells and examining EphB4 expression, where detection of increased EphB4 expression compared to normal esophageal samples indicates the presence of an esophageal condition. Provided is a method for diagnosing the condition of the esophagus. In certain embodiments, the condition of the esophagus is selected from the group consisting of Barrett's esophagus and esophageal cancer. The diagnostic method is also envisioned to include monitoring EphB4 expression in patients. For example, such monitoring may involve repeated sampling of patient samples and measurement of EphB4 expression as a method of monitoring the patient for the presence and / or progression of the disease. The present invention is also a kit for performing such methods, an anti-EphB4 antibody and / or EphB4 for detecting the expression of the EphB4 protein. A kit containing PCR primers for detecting mRNA expression is provided. One such kit envisioned in the present invention is an ELISA-based kit that may allow quantitative or qualitative measurement or comparison of EphB4 protein levels in a sample. Various immunological assays can be readily used by those skilled in the art to detect expression of the EphB4 protein. Several companies sell antibodies that can be used to specifically detect EphB4 in esophageal samples. Such methods for making measurements of EphB4 levels may include, but are not limited to, immunohistochemical and concentration measurements of Western blots used to confirm the presence or absence of EphB4. Those skilled in the art will be able to recognize and use alternative methods such as, but not limited to, ELISA and real-time RT-PCR. The examples below show the use of PCR to detect EphB4 expression. Those skilled in the art will be able to readily prepare primers suitable for detecting EphB4 expression using standard techniques.

Expression of EphB4 on normal GI epithelial cells is very low, combined with confirmation of changes in the conformation of EphB4 receptors in cancer cells when compared to healthy cells, in mammals or birds. The chances of safe and effective vaccination, as well as the safe and effective cancer prevention and / or treatment with the milk or egg antibody, are increased. Also, healthy intestinal epithelial cells generally express EphB4 only below tight junctions where locally administered antibodies are not reachable. For this reason, antibodies applied topically to cell surface molecules have a much safer profile than systemically administered monoclonal antibodies.

The present invention relates to non-infectious medical conditions. In contrast to infectious conditions, non-infectious medical conditions are generally established in or in patients. As established, non-infectious medical conditions are less sensitive to treatment with pass-through agents and are therefore more difficult to treat than infectious conditions, including infectious entities that are not normally adherent to the patient. Is. Infectious agents are generally free "organisms" such as bacteria or viruses and are susceptible to treatment with, for example, antibacterial or antiviral agents. The present invention relates to the prevention and / or treatment of more difficult non-infectious medical conditions without the assistance of systemic factors commonly provided by the immune system.

As described above, in order to produce milk having a high affinity and selective antibody to the target molecule, it is preferable to vaccinate the mammal with an appropriate antigen. There are several possible strategies for delivering the antigen at the appropriate purity and amount. The selection of such strategies is described below. However, it should be noted that such strategies and target proteins are exemplary, and those skilled in the art will appreciate additional strategies or atypical forms for those described below. In addition, EphB4 is used as a mere example of a molecule to which such a method is applicable. Those skilled in the art will be able to apply such methods and the like to other latent target molecules.

Pure ephrin-B1 and / or B2 are required for affinity purification of EphB4 by binding to the ligand. The native ephrin B1 ligand can be purified from suitable cells using standard biochemical protein purification techniques, such as, but not limited to, ion or cation exchange chromatography and size exclusion chromatography. Alternatively, ephrin can be affinity purified by the use of anti-ephrin antibodies. One approach is to prepare a specific immunoaffinity column and purify ephrin-B1 / B2. A further alternative approach is to use recombinant means to produce large amounts of ephrin-B1 / B2. The procedure for doing this is publicly available. This The advantage of this approach is that all that is needed is the creation of the Eph binding region of the molecule. Once purified, immobilization of the ephrin or ephrin fragment on the support may allow affinity purification of EphB4 from cell lysates by standard procedures.

Such a methodology can result in a complete EphB4 with both extracellular and intracellular parts of the molecule. However, only the extracellular portion of the molecule is relevant as an antigen. The EphB4 molecule is about 120 kDa in size and the extracellular portion of the molecule is about 50 kDa.

An alternative approach for producing large amounts of EphB4 is to use recombinant means. As with ephrins (s), all that is needed is the production of extracellular components of the molecule. As mentioned above, the procedure for doing this is publicly available.

Vaccination of mammals or birds with large molecules may not be the most efficient method of producing highly specific antibodies with the desired therapeutic effect. Therefore, in order to identify the most suitable antigen for vaccination of mammals or birds, it is necessary to fragment EphB4 or other target molecules and test the antibody generated against the fragment for its therapeutic effect. Is.

For example, several insolubilized proteolytic enzymes can be used to systematically degrade (hydrolyze) the EphB4 molecule (or its extracellular domain). Exemplary enzymes include, but are not limited to, trypsin, pepsin, papain and bromelain. Hydrolysis of EphB4 with a proteolytic enzyme can yield a variety of peptides, and the use of the enzymes individually and in combination can allow the formation of overlapping fragments.

One method for making antibodies against each of the fragments is to purify each fragment individually and immunotreat the test animal with each fragment. Alternatively, a single animal may be vaccinated with multiple fragments, the resulting antibody may be purified from immune sera by affinity chromatography with individual peptides, where the appropriate antibody is available. , Binds to its respective peptide.

It is envisioned that standard laboratory animals can be used in this antigen identification step. For example, mice or rabbits can be used to generate polyclonal antibodies using EphB4 fragments. Standard protocols for vaccination and antibody purification can be used.

The resulting antiserum can be tested, for example, in a human colon cancer cell line in an in vitro cell culture assay to detect the antitumor effect of the antibody on each of the EphB4 fragments. Once the antitumor effect is confirmed, the antibody binding fragment (s) that provide the effect can be identified.

EphB4 fragments or other peptides that form part of a target molecule with antitumor or other therapeutic effects are in large quantities using standard procedures, such as, but not limited to, recombination techniques or peptide preparation and conjugation. Can be generated in. Such fragments can then be used as immunogenic peptides (vaccine antigens) to produce anti-EphB4 or other specific antibody-containing milk.

Immunogenic peptides are assumed to be at least 10 amino acid residue lengths, whether for EphB4 or other target molecules. In other forms of the invention, the immunogenic peptide can be at least 20, 30, 40, 50, 60 or 100 residue length. In a further form of the invention, the immunogenic peptide can be the domain of the protein, eg, the extracellular domain of the protein. In certain embodiments of the invention, the length, composition and conformation of immunogenic peptides are naturally occurring between mammals or birds and patients receiving antibody-containing milk or egg products. It can be determined by the degree of sequence homology of the protein.

The cost of peptide vaccines is probably very low when compared to viral or bacterial vaccines used for infectious diseases. This may have some advantages, but not limited to, over conventional treatment modalities and with respect to the production of therapeutic antibodies. 1. Topical treatment with locally applied polyclonal antibody-containing milk products or egg products has both cost and safety advantages over systemically applied monoclonal antibodies; and 2. The present invention may solve the problems associated with antibody-containing milk products or egg products that have been shown to be very expensive to produce. The production of monoclonal antibodies for use in systemic treatment regimens is very expensive compared to the production of antibody-containing milk products or egg products. In addition, in the methods of the invention, one of a number of animals that yields milk or egg products can be used, which produces highly specific and active antibodies against any potential antigen. Opportunities can be significantly better.

The choice of mammal or bird for use in the methods of the invention depends on several factors, such as the amount of antibody-containing milk or egg product required, and the selected immunogenic protein or peptide. Determined by the level of immune response that can result from. The level of immune response resulting may reflect the degree of sequence homology between the immunogenic protein or peptide and its corresponding sequence in a mammal or bird. Mammals or birds probably do not exhibit an immune response to "self-antigens." That is because this would be an effective initiation of autoimmune disease. If such a situation occurs, it can clearly be harmful to the mammal or bird in question. Therefore, the present invention envisions the requirement to use a variety of species to identify the species that provide the required immune response.

It should also be noted that there may or may not be a difference between the same target molecule expressed by healthy and diseased cells. If there is a difference, the immunogenic peptide incorporating this difference can be used to provide an immune response directed to a non-wild-type antigen found only in the molecule of the diseased cell. Careful immunogenic selection is required if diseased cells simply exhibit higher levels of the exact same target antigen than normal cells. For example, in some GI tumors, there is a difference between EphB4 expressed in cancer cells and healthy cells. Thus, antibodies for use in the methods of the invention may be generated against EphB4 peptides that are not shown in either sequence or structure in healthy cell EphB4. If EphB4 is simply upregulated, it may be necessary to carefully monitor antibody administration to avoid potentially harmful side effects. The advantage provided by the present invention is that the antibody is administered topically, which allows the area where the diseased cells are present to be targeted, which dramatically reduces the amount or severity of side effects. , It has the potential to provide greater safety than the systemic approach in which monoclonal antibodies are used.

Immunogenic peptides can be synthesized by the use of peptide synthesizers. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the polypeptide sequence as substitutions or additions. Non-classical amino acids include, but are not limited to, common amino acids, ornithine, norleucine, norvaline, hydroxyproline, sarcosin, citrulline, homocitrulline, cysteine acid, t-butylglycine, t-butylalanine, fu. Examples include D-isomers of enylglycine, cyclohexylalanine and β-alanine.

If desired, various techniques can be used to develop greater antibody reactivity in order to increase the resulting immune response of the immunogenic peptide. For example, the desired peptide may be coupled to another carrier protein, such as, but not limited to, ovalbumin or scabigai hemocyanin (KLH), and / or an adjuvant, such as, but not limited to, Freund's complete or incomplete adjuvant. Can be used.

One of the exemplary immunogenic peptides that can be used in the methods of the invention is PVAGSCVVDAVPAPGPSPSLY (SEQ ID NO: 4). This 21 amino acid sequence represents a fragment of the extracellular domain of human EphB4 from cancerous cells. It is envisioned that shorter, longer, or different immunogenic peptides could be prepared for EphB4, and some such alternatives are described in WO2004024773.

It was found that the sequence shown in SEQ ID NO: 4 differs from the corresponding bovine sequence (PVAGSCVADAMPAPGPSPSLY, SEQ ID NO: 5) only in two positions. Thus, in one aspect of the invention, the polyclonal anti-EphB4 antibody against the EphB4 peptide may have been produced in avian species and the resulting polyclonal IgY antibody is a cancer or condition characterized by upregulation of the EphB4 protein. Can be used for non-systemic prevention and / or treatment of. One such condition is Barrett's esophagus, which is discussed herein and shown in the Examples.

The above discussion indicates that a single immunogenic peptide can be used to vaccinate a mammal or bird. Note that more than one immunogenic peptide may be used. More than one immunogenic peptide can be administered simultaneously or sequentially. In addition, one or more immunogenic peptides can present the same or different molecules. When one or more immunogenic peptides exhibit the same molecule, different immunogenic peptides can result in a more robust immunogenic response to one molecule or variant of one molecule. If one or more immunogenic peptides exhibit different molecules, this will allow antibody-containing milk or egg products to prevent and / or treat non-communicable medical conditions as described herein. When used, a broader approach to inhibition of target cell proliferation may be provided.

As described above, it is also possible to use the method of the present invention in mammals other than cows to produce antibody-containing milk products. It may be necessary to use such other mammals, especially if the cow is unable to exhibit a valid immune response against the desired immunogen. This can occur if the bovine and the patient to be treated have high sequence homology of the immunogenic peptide. Other mammals such as sheep, pigs and goats can typically be used. Antibody-containing milk is not intended to be used on the same scale as regular milk, thus reducing the requirements for production on a very large scale. Therefore, such an alternative milk source may be useful. It is convenient to produce antibody-containing egg products with or instead of antibody-containing milk products because of heterogeneity (heterology) and / or patient preference and / or economics that result in an increased immune response. possible.

Milk products and / or egg products that contain antibodies to more than one specific molecule can be vaccinated against a single mammal or bird with multiple antigens, or with antigens against various target molecules. Can be produced by mixing the milk and / or eggs of a mammal or bird that has been vaccinated.

Once the immune response is initiated in a mammal or bird, it is desirable to collect as much antibody-containing milk or eggs as possible. Typically, the first milk contains high levels of antibody, but once normal milk production begins, the level of antibody generally decreases. Therefore, it would be desirable to collect only the first milk in order to obtain the antibody. However, the use of various vaccination methods allows relatively high levels of antibody to be retained in the milk for extended periods of time after the end of primary milk production. Such methods are particularly useful in the present invention for producing high volume final products suitable for nutritional products and pharmaceuticals.

In certain embodiments of the invention, mammals such as commercial dairy cows can be vaccinated intramuscularly with the buttocks. After the first immunostimulation, the mammal is delivered with the maximum amount of specific antibody in the first milk and boosted to maintain the specific antibody level in the milk during milk production. Is expected.

In the case of birds (such as chickens, geese and ducks), they are first immune stimulated at any time and boosted at various times to maximize antibody levels in the egg over a long period of time. ..

Once an antibody-containing milk or egg product is obtained, it is generally (but not always) necessary to process the product into a form suitable for administration. It is important that the antibody is not adversely affected by any treatment. Some such methods require cold heating to preserve the functionality of the antibody. In addition, it is desirable that the antibody remain protected from the proteolytic effects of the animal or person to which it is administered.

In the present specification, an exemplary embodiment using an antibody-containing milk product will be described later. One of the exemplary antibody-containing milk products is bovine first milk. Cow colostrum is secured in the first 4 days after birth, more preferably cow colostrum is secured in the first 2 days after birth, and even more preferably cow colostrum is secured on the first day after birth. Most preferably, bovine colostrum is the first milking after calving.

In a particular embodiment of the invention, the antibody-containing milk product can be processed using a degreasing operation. In other embodiments, the processing further includes an operation for removing cell debris. In yet a further embodiment, the processing may further include an operation for removing salts, sugars, other low molecular weight entities and some water.

In a particular embodiment of the invention, the antibody-containing milk product comprises at least 4% total protein (% by weight). In other embodiments, the protein content can be at least 50%, or at least 70%, as well as at least 80%.

The antibody content of the antibody-containing milk product can also be measured. Depending on the end use of the product, IgG can be any as a percentage of the final formulation, from approximately 2% of the Sippy dietary formulation to over 90% for affinity purified topical formulations.

The antibody-containing milk product is expected to contain the above antibodies or fragments thereof, such as monoclonal or polyclonal immunoglobulins. The product is a chimeric monoclonal antibody or a humanized monoclonal antibody or a dendrimer-presented immunoreactive fragment or an immunoreactive fragment (F (ab) and F (ab)).₂Fragments, etc.) or recombinant immunoreactive fragments, or affinity purified immunoglobulins or immunoreactive fragments thereof. To those skilled in the art, some of the above antibodies cannot be produced by immunotreated mammals or birds, but such antibodies are added to milk or egg products prior to administration to a patient. It will be self-evident to get.

It is envisioned that antibody-containing eggs produced in accordance with the present invention can be processed using standard methods known in the art. Some such methods require cold heating to preserve the functionality of the antibody. It is also assumed that the specific antibody can be isolated from the egg by a technique such as affinity chromatography.

The milk or egg products of the present invention can be treated by several methods to eliminate contaminating bacteria and to extend shelf life. Such methods include, but are not limited to, microfiltration.

The compositions of the present invention are, but are not limited to, capsules, powder tablets, aerosol sprays, syrups, solutions, suppositories, enema or other forms known in the art that are easy to administer non-systemically. It can be administered in a series of forms such as. The composition may further include carriers or excipients suitable for gastrointestinal or other topical administration. Examples of carriers and excipients include silica, talc, titanium dioxide, alumina, starch, kaolin, powdered cellulose, microcrystalline cellulose, amylopectin N, sucrose, lactose, dextrose, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, hydroxy. Examples thereof include ethyl cellulose, carboxymethyl cellulose, citric acid, sodium bicarbonate, magnesium stearate, shelac, cellulose acetate, cetyl alcohol, triethyl citrate and polyethylene glycol.

Also, the preparation of nutritional supplements or pharmaceuticals may require the formulation of antibody-containing milk products or egg products into administrable forms. For example, tablets or suppositories containing antibody-containing milk products or egg products may require a coating to make them suitable for swallowing or insertion, as needed. Such a coating may contain a protective material that prevents the encapsulated product from being inactivated, eg, in the stomach, but allows the release of the product in the patient's intestine. Such coatings and protective materials are known in the art.

In addition, the nutritional supplement product using the antibody-containing milk product or egg product may contain a preparation having additional nutritional components. Such additional nutritional components may have beneficial medical effects for the patient being treated. Other components are not particularly limited and include, for example, amino acids, vitamins, minerals, prebiotics, probiotics and other functional components.

Amino acids for inclusion in nutritional products may be selected from the group comprising glutamine, glutamic acid, tryptophan, alanine, arginine, aspartic acid, threonine, serine, gamma-aminobutyric acid, taurine, thiotaurine, hypotaurine.

Vitamins to be included in nutritional products are not limited, but vitamin A, vitamin B₁, Vitamin B₂, Vitamin B₆, Vitamin B₁₂, Vitamin C, Vitamin D, Vitamin E, Vitamin K, Folic Acid, Nicotinic Acid, Lipoic Acid, Pantothenic Acid, Biotin, Ubiquinone, Prostaglandin and derivatives of these vitamins can be selected from the group.

Minerals for inclusion in nutritional products may be selected from the group containing, but not limited to, calcium, iron, magnesium, copper, zinc, selenium and potassium.

Prebiotics for inclusion in nutritional products can be selected from the group comprising fructooligosaccharides (FOS), galactooligosaccharides (GOS), acidic oligosaccharides (AOS) and inulin.

Other functional substances to be included in nutritional products are, but are not limited to, ginseng, herbs (chamomile, gymnema / garcinia, etc.), Chinese herbal plants (such as Tochu and Korean ginseng) or their extracts, extracts of animal origin. It can be selected from the group containing (such as placenta), dietary fiber (such as oligosaccharides and polysaccharides), soybean peptides, diet sweeteners and caffeine.

The milk product or egg product of the present invention can be in the form of a nutritional composition. Such compositions may include 10-60en% lipids, 5-50en% proteins, 15-90en% carbohydrates. The amount of each component in the composition is measured in "en%", which unit is the "energy percentage" and represents the relative amount that each component contributes to the total caloric value of the preparation. Certain nutritional compositions may contain 7.5 to 12.5 en% protein; 40 to 55 en% carbohydrates; and 35 to 50 en% fat.

In addition, at least one long chain selected from the group consisting of eicosapentaenoic acid (EPA, n-3), docosahexaenoic acid (DHA, n-3) and arachidonic acid (AA, n-6) in the composition. Polyunsaturated fatty acids (LC-PUFA) may be included because they further reduce airway infections and / or their symptoms. The compositions of the present invention may also contain AA and DHA and optionally EPA.

The composition may also include LC-PUFAs having a total fat content of at least 0.1 wt%, at least 0.25 wt%, at least 0.5 wt%, or at least 0.75 wt% and having 20 to 22 carbon atoms. The content of LC-PUFA having 20 to 22 carbon atoms in the composition of the present invention should not exceed 15 wt% of the total fat content, and optionally, the content is 10 wt% of the total fat content. It is better not to exceed%, and it is better not to exceed 5wt% by arbitrary choice.

The EPA content of the composition should not exceed 15 wt% of total fat, optionally, the content should not exceed 5 wt%, and optionally should not exceed 1 wt%. However, the EPA content can be at least 0.05 wt%, or at least 0.1 wt%, of total fat. The DHA content should not exceed 10 wt%, optionally, the content should not exceed 5 wt%, and optionally should not exceed 1 wt%. However, the DHA content can be at least 0.1 wt% of total fat. The compositions of the present invention may contain at least 0.1 wt% AA, at least 0.25 wt% AA, or at least 0.5 wt% AA with respect to total fat. The AA content should not exceed 5 wt% of total fat and, optionally, not more than 1 wt%.

Formulations known in the art suitable for delivery to the bladder via a catheter, delivery to the eye with eye drops or ointments, delivery to the oral cavity / oral vestibule with patches or gums, etc. may be used.

The milk or egg products of the present invention can also be used in the treatment and / or prevention of skin cancer or hyperproliferative conditions of the skin. In a particular embodiment of the invention, the milk product or egg product may be included in cosmetics and / or pharmaceutical topical formulations suitable for skin application.

It is envisioned that the cosmetic formulations according to the invention provide therapeutically effective doses, maintain the stability of the antibody over time, and allow contact between the antibody and the target molecule. High temperature heating should be avoided in the formulation process to preserve the functionality of the antibody. Furthermore, it is envisioned that cosmetic formulations may be in the form of creams, ointments, drops, sprays, patches, mask-like formulations and the like.

In a second aspect, the invention provides a method of preventing and / or treating a patient's non-communicable medical condition, comprising administering to the patient an antibody-containing milk product or egg product in a non-systemic manner. To do. In one embodiment of the invention, the milk product or egg product contains an antibody directed to an immunogenic molecule of mammalian origin. In other forms of the invention, mammalian-derived immunogenic molecules can be found on the surface of mammalian cells (such as cancer cells). In a further form of the invention, the mammalian-derived immunogenic molecule can be a mutant protein or a protein normally not found on the surface of mammalian cells.

The methods and products of the present invention may assist in the treatment as well as the prevention of GI ducts (including the oropharynx), eyes, ears, nasopharynx, bladder, vagina, uterus, neck and skin conditions. is assumed. Prevention is due to the fact that the antibody targets only molecules found on cancerous cells or molecules that are upregulated in the cells, and that the body has a barrier to the systemic arrival of intact antibodies. Be assisted.

It is assumed that the method of the present invention can assist in the prevention of cancer formation in individuals in the risk group. Antibodies in antibody-containing milk products or egg products bind to cells overexpressing the protein or molecule to which the antibody is directed, followed by inhibition of cell growth. Thus, antibody-containing milk or egg products can provide, for example, nutritional support and prophylactic effects to persons at risk for colorectal cancer or other GI cancers and conditions, or skin or other. Applications may be found in cancer-preventing cosmetics for organ application, or in pharmaceutical formulations for application to the skin, eyes, oropharynx, uterus, vagina / neck or bladder.

Topical treatment of corneal cancer can be the most effective means of delivering eye-effective treatments, is immunologically inactive, and is detrimental to the topical application of foreign proteins envisioned in this application. Not affected.

Current treatments for skin cancer typically involve surgery to remove the cancer. The approach of the present invention is significantly simpler than surgery to remove internal cancer. However, there is a need to prevent skin cancer formation, which is a role that can be played by the milk or egg products of the present invention. It is envisioned that the compositions incorporating the milk or egg products of the present invention may be safely applied to the skin. Antibodies in milk or egg products can bind to cells that express molecules associated with skin cancer on their surface. This event then causes the cells to destroy before they can develop into cancer. In addition, such compositions may find application applications in reducing the likelihood of postoperative skin cancer re-expression.

In addition, the method of the present invention may find application in the treatment of GI cancer. In one embodiment of the invention, the antibody in the antibody-containing milk product or egg product binds to cells overexpressing the protein to which the antibody is directed, followed by inhibition of proliferation of the cells. Is done. For such an action, a use as a therapeutic agent for GI cancer can be found. In another embodiment of the invention, oral ingestion of antibody-containing milk or egg products can be used as a further treatment to supplement conventional treatment of GI cancer. It is envisioned that antibody-containing milk or egg products may increase the likelihood of remission of a patient's GI cancer and may increase the duration of remission. Therefore, the method of the present invention can improve the annual survival rate of GI cancer patients.

Current treatments for colorectal cancer include surgical resection of the disease-free margin of 3-5 cm and resection of the mesentery, which is the source of blood supply and contains the major lymphatic drainage site. Invasive treatment is required to attempt to cure the disease. Usually, artificial anus plasty can be avoided unless the cancer is within 5 cm of the anus.

Cure can be achieved with surgery alone in a large number of patients. If cancer is detected in the early stages, the rate of cure will be much faster. Overall, nearly 75% of patients are cured by primary resection, 25% of patients relapse, and 20% of them are cured by secondary resection.

Chemotherapy is also standard for most patients. It generally involves a combination of 5-fluorouracil (5-FU) and levamisole for a period of 1 year after surgery and can be used in combination with radiation therapy. Chemotherapy with 5-FU and levamisole has been shown to increase patient overall survival from 46% to 60% after 6.5 years compared to patients treated by surgery alone.

Survival of esophageal cancer is generally low as a result of patients frequently suffering from swallowing and / or feeding problems, which can lead to malnutrition. In certain embodiments of the invention, the antibody-containing milk product or egg product can be administered directly in the vicinity of esophageal cancer in the GI lumen. In addition, as described herein, the product may contain nutritional components as well as antibodies to the target cells, which may assist in alleviating some of the symptoms associated with malnutrition. ..

In the case of GI status, the method of the invention administers an antibody-containing milk product or egg product to a patient via the GI lumen, whereby the antibody in the milk product contacts cancer cells in the GI duct. Includes steps. Antibody binding can be rapid and long lasting, so even a single dose of antibody can have a significant short-term effect on the condition. Continuous dosing can be expected to have a significant long-term effect on the condition. Exemplary administration methods of antibody-containing milk products or egg products include, but are not limited to. -Drinking antibody-containing milk products or egg products. As a result, it passes through the GI tube, whereby the antibody in the milk or egg product binds to the target antigen / molecule during passage. A suitable composition for this approach is 1) Protective compounds and / or 2) Combined antacid and / or 3) Viscous composition that is ingested little by little over time It is assumed that the antibody in the milk product or egg product may survive the digestive process in the GI tube (particularly in the esophagus) or remain in the GI tube for a long period of time. It helps to prolong the contact time between the antibody and the target molecule. -Direct introduction of antibody-containing milk or egg products into the esophagus or intestine (large intestine or small intestine). This is a pump / tube set by injecting or pumping an antibody-containing milk product or egg product through a tube into the required portion of the GI tube, or over the area where treatment is required. Can be done by spraying directly through Is mentioned, In the treatment of esophageal conditions, balloons placed near the stomach may be used to prevent antibody-containing milk or egg products from entering the stomach too quickly, optionally. It is envisioned that additional balloons placed in the upper part of the esophagus could be used to prevent backflow of antibody-containing milk products or (ore) egg products. In this way, long-term treatment of affected or risk areas can be achieved with non-large amounts of antibody-containing milk or egg products. In the treatment of colonic conditions, antibody-containing milk products can be introduced directly into the colon in the form of enema, by colonic irrigation, or in solid or semi-solid form as suppositories.

It is envisioned that the methods and products of the present invention may reduce the incidence of GI cancer in the risk group, delay the onset of GI cancer in the risk group, and increase the rate and duration of remission in patients with GI cancer. To. As a result of these effects, it is expected that the annual survival rate will increase at the same time in the risk group and the patient group.

In skin conditions, the method comprises the step of administering the antibody-containing milk product or egg product directly to the patient's skin, thereby contacting the antibody in the milk product or egg product with the target cells.

It is assumed that the incorporation of the product of the present invention into cosmetics can also assist in reducing the incidence of skin cancer or preventing skin cancer. Many cosmetics now include sunscreens to reduce the potential for sun damage to the skin. By incorporating the products of the present invention, cosmetics can also assist in the restoration of damage to the skin that has already been inflicted, thereby preventing the formation of skin cancer.

For other topical applications (eyes, ears, nose, throat, vagina, neck, bladder), direct application of the appropriate pharmaceutical formulation is preferred.

Topical treatment of corneal cancer can be the most effective means of delivering eye-effective treatments, is immunologically inactive, and is detrimental to the topical application of foreign proteins envisioned in this application. Not affected.

In a further aspect, the invention provides an antibody isolated from an antibody-containing milk product or egg product of the invention. Such antibodies can be isolated using standard methods known to those of skill in the art. Such methods include, but are not limited to, affinity purification by binding of the antibody to the immobilized antigen by means of affinity chromatography.

The present invention also provides a pharmaceutical composition comprising an antibody-containing milk product or egg product of the present invention and / or an antibody of the present invention. The composition may also contain various pharmaceutically acceptable ingredients that make the composition suitable for administration to a patient. The various ingredients include, but are not limited to, pharmaceutically acceptable diluents and buffers. As mentioned above, there are various routes for administration according to the present invention. One of ordinary skill in the art will be able to formulate suitable compositions for various routes of administration.

In a further aspect, the invention provides a kit that can be used to diagnose and / or treat and / or prevent a patient's non-infectious medical condition. Such kits include antibody-containing milk or egg products of the invention and / or antibodies of the invention and / or pharmaceutical compositions of the invention. The kit can be used to diagnose and / or treat and / or prevent non-infectious medical conditions, such as cancer (such as gastrointestinal or skin cancer). In certain embodiments of the invention, the kit comprises anal cancer, bile duct cancer, colon-rectal (colon and / or rectal) cancer, esophageal cancer, gallbladder cancer, gastric cancer, liver cancer (hepatoma), pancreatic cancer and small intestine. Can be used for the diagnosis and / or treatment and / or prevention of gastrointestinal cancers that are selected from the group consisting of cancers.

Next, specific embodiments and applications of the present invention will be discussed in detail with reference to the examples described later. This article is not intended to limit the scope of the invention in any way.

<p> Example 1. Identification of cell surface target molecule Samples from healthy donors and patients with esophageal adenocarcinoma were collected from excess tissue remaining at biopsy during routine endoscopy or after esophagectomy. Patient-derived control tissue was taken from the endoscopically visualized area of normal squamous epithelium 2-5 cm proximal to the tumor margin. Tissues were immediately stored in RNA Later or RNA Later Ice . Histopathologists identified a small section of the tissue used for RNA extraction as normal squamous epithelium (N), or Barrett epithelium (intestinal metaplasia, IM), or adenocarcinoma (tumor, T).</p><p> RNA was extracted using Trizol and reverse transcribed into cDNA using Superscript III . The final volume of cDNA was 400 μl. 6 μl of cDNA in 20 μl of PCR reaction solution (1 × Quantitect Sybr Used in Green real-time PCR mixes and 200 nM forward and reverse primers, respectively). The PCR reaction was performed on a Corbett Rotorgene real-time PCR device (model 3000 or 6000). Gene-specific PCR signals were standardized with β-actin. We report the quantification of Eph and ephrin for colon adenocarcinoma cell line HT29 set to 1 at our discretion.</p><p> The data were evaluated for statistical significance using the nonparametric Clascal Wallis test. Multiple comparisons were made by the Clascal-Wallis test, followed by Bonferroni correction. A p value less than 0.017 was considered significant.</p><p> The study included 11 patients with esophageal adenocarcinoma. Figures 1 and 2 show the relative mRNA levels of Eph and Efn in tissues from these patients and healthy donors. The data is shown in a box plot and the whiskers represent the range of data points. The three histologically identified histological types (N, IM and T) are shown in separate box plots. All of the analyzed Eph and Efn showed significant differences in mRNA levels between normal tissue vs. bullet tissue and normal tissue vs. esophageal adenocarcinoma tissue.</p><p> In general, the mRNA levels of both evaluated EphA receptors were significantly lower in IM and T compared to N, whereas the mRNA levels of all EphB receptors examined were significantly lower in IM and T compared to N. it was high. Both EfnA1 and EfnB2 mRNA levels were significantly higher in IM and T compared to N. Table 1 summarizes the median level change multiples of mRNA for each of these genes in various tissue groups.</p><p> Table 1: Multiples of changes in Eph and Efn median mRNA levels in IM and T tissues relative to N tissue</p><p><tables num="1"><img file="JP2010501596A_D0001.tif" /></tables></p><p> Comparing normal tissues with IM and T groups, EphB2 mRNA levels have a maximum increase in median mRNA levels (80-100 fold) and EphA1 has a maximum decrease in median mRNA levels (3-4 fold). ). Esophageal adenocarcinoma showed a tendency to decrease median EphB2 and EfnB2 mRNA levels compared to Barrett's epithelium. In contrast, median EphB4 mRNA levels tended to increase in esophageal adenocarcinoma compared to Barrett's epithelium. Any gene that exhibits upregulation in IM and / or T can be a potential target for antibody production and therapeutic instructions described herein.</p><p> Significant increases in EphB4 mRNA levels in adenocarcinoma when compared to normal tissue are slower than those in EphB2, but are interesting because of the association between increased EphB4 levels and cancer cell survival. The tendency for EphB4 mRNA to increase in esophageal adenocarcinoma compared to Barrett's epithelium may be associated with the acquisition of a transforming invasive cancer phenotype.</p>

<p> Example 2. EphB4 expression in an esophageal cell line The expression outlined in Example 1 above was used to examine the expression of EphB4 mRNA in Het1A and OE33 cell lines. Het-1A cells are derived from normal esophageal epithelium and OE33 is derived from esophageal adenocarcinoma epithelium. Four cultures of Het-1A and three cultures of OE-33 were examined. The results shown in FIG. 3 show that EphB4 expression is significantly higher in the adenocarcinoma-derived cell line (OE33) than in the essentially normal Het1A cell line.</p>

<p> Example 3. Preparation of EphB4 immunogen Several procedures can be used to obtain a suitably pure EphB4 antigen. In one method, a pure ephrin B1 (or B2) ligand is used to covalently attach (efrin-B1, B2) to a suitable chromatography matrix, which is the EphB4 receptor from a crude (colon tumor) cell extract. Purify the EphB4 receptor by using it for affinity purification. Alternatively, the EphB4 receptor is purified using immunoaffinity purification using a specific polyclonal anti-EphB4 antibody covalently attached to a suitable chromatography matrix. Alternatively, EphB4 is produced by recombinant techniques according to published procedures.</p><p> Standard biochemical protein purification techniques (eg, ion or cation exchange chromatography, size exclusion chromatography, etc.) were used as possible methods for producing pure ephrin-B1 (B2) -ligands. Isolation of the native ephrin-B1 ligand from suitable human cells can be mentioned. The second method is immunoaffinity purification of the ephrin-B1 ligand. In either case, final purification can be performed using a commercially available polyclonal anti-ephrin B1 (B2) ligand antiserum. A third alternative is the production of recombinant ephrin-B1 ligands. This has the advantage that a large number of molecules can be produced in a short time. Also, all that is needed is the preparation of the extracellular (Eph receptor binding) portion of the molecule.</p><p> As mentioned above, one of the options for preparing the EphB4 antigen is an affinity purification method using an ephrin-B1 ligand prepared as described above from a natural source such as various types of colon cancer cells. Is to be isolated by. This procedure results in a complete EphB4 receptor with both extracellular and intracellular parts of the molecule.</p><p> Since only the extracellular portion of the molecule (approximately 50 kDa out of a total of 120 kDa) is relevant as an antigen, an alternative approach to the preparation of the EphB4 molecule is a recombinant methodology for preparing only the extracellular domain. It is used. Recombinant methodologies have the advantage of allowing large-scale preparation of specific molecules. However, the potential drawback is that post-translational modifications are generally not reproduced in recombinantly produced molecules. Purified proteins may be a better approach if modified residues in the extracellular domain are important in eliciting an immune response to EphB4.</p><p> Several insolubilized proteolytic enzymes can be used to systematically degrade (hydrolyze) the EphB4 receptor. The enzymes used are trypsin, Examples include pepsin, papain and bromelain. Hydrolysis of the EphB4 receptor by proteolytic enzymes yields a variety of peptides derived from both the intracellular and extracellular components of the molecule. Purification of these peptides yields individual EphB4 peptides. Because size exclusion chromatography separates peptides based solely on size differences, different peptides of the same size are eluted in the same fraction. However, if these peptides are known to be good antigens, they can be further separated by ion exchange (or other) chromatography.</p><p> Limited hydrolysis of EphB4 can be achieved by using insolubilized proteolytic enzymes (trypsin, pepsin, etc.). This results in a small number of large EphB4 protein fragments (20-40 kDa in size). Such protein fragments are large enough to elicit a good antibody response in immunotreated animals. In addition, such large protein fragments retain local protein surface topography (tertiary structure). This can ensure that the antibody produced is against an epitope present on the native EphB4 protein.</p>

<p> Example 4. Identification of EphB4 antigen It is necessary to establish which EphB4 peptide fragment produces a high affinity EphB4 antibody when used as an antigen. To achieve this, some rabbits (eg) can be immunotreated with the peptides described above and the resulting antisera can be tested in an in vitro cell culture assay using human colon cancer cell lines.</p><p> As mentioned above, only peptide fragments derived from the extracellular portion of the EphB4 receptor can be used as effective antigens. Only this part of the molecule can reach the extracellular antibody, and the ephrin ligand binding site is located on this part of the molecule, blocking it to stop EphB4 signaling.</p><p> Rabbits can be immunotreated with the complete (undigested) EphB4 receptor. This rabbit-derived antiserum can be provided as a "control serum" to which another antiserum can be tested in contrast. Rabbits may also be immunotreated with an EphB4 peptide fragment (any animal in this group can be immunotreated with a mixture containing all of the peptide fragments).</p><p> Animals other than rabbits, such as mice, rats, and guinea pigs, may be used for immune treatment. However, small animals are difficult to immunize and collect blood, and the amount of antiserum that can be recovered is very limited.</p><p> It may be possible to immunize rabbits with individual peptide fragments to obtain antisera specific for each particular EphB4 peptide, but for the preparation of pure fragments and the immunization of several animals per fragment. Can involve a large amount of work. Immune treatment with a mixture of fragments is efficient. After approximately 12 weeks, crude rabbit antisera can be tested for efficacy in an in vitro cell culture assay using human colon tumor cells. All that is required for such an assay is some indication of inhibition of tumor cell growth and growth. Once positive antitumor effects are shown, anti-EphB4 antibody is purified from hyperimmune rabbit serum to identify the most effective antibody. This is done by preparing a small affinity column for each individual EphB4 peptide fragment used as an antigen. When crude rabbit serum is passed through a column, the appropriate antibody here (were) binds to its respective peptide antigen. In this way, all anti-EphB4 (peptide fragment) antibodies are separated from crude rabbit serum. Specific EphB4 peptide antibodies are then tested (individually) in an in vitro cell culture assay using human colon cancer cell lines. In this way, the most effective antigen, the EphB4 peptide fragment Can be identified. When such peptides are then used as immunogens (antigens for vaccination), anti-EphB4 antibody-containing milk products are produced.</p><p> Although it may be possible to identify immunogenic EphB4 protein fragments using commercially available α-EphB4 polyclonal antibodies, the problem with this procedure is that this antibody reacts well with abnormal / tumor-derived EphB4 receptors. It may not be. The most important antigens are these cancer / tumor-derived abnormal (mutant) EphB4 receptors. With the use of commercially available antibodies, it is possible that biologically significant antigens may be overlooked.</p><p> The amino acid sequence of such a fragment can be determined, from which a method of its production by recombinant means can be established. This makes it possible to produce an infinite amount of antigen. The antigen can provide an effective anti-EphB4 antibody with high affinity.</p><p> Subsequent patent applications may be filed in Australia or abroad on the basis of this application, for example by claiming priority of this application, filing a divisional application and / or filing a continuation application. The appended claims are provided for illustrative purposes only and are not intended to limit the scope of claims that can be claimed in any such subsequent application. It will also be appreciated that the claims should not be deemed to limit (or exclude) the understanding of the invention (s) inherent in the present disclosure. Features may be added to or removed from the examples of the claims at a later date, as further defined by the present invention (s).</p>

<p> Example 5. Peptide production and conjugation Synthesis of peptides for use in the methods of the invention can be performed by standard methods known to those of skill in the art. In practice, amino acid residues are continuously added to the immobilized amino acids by an automated process. Once an amino acid has been added, it is then deprotected to accept the next amino acid residue. Amino acid residues are protected to ensure that only one residue is added per reaction step and that the side chains of the amino acid residues are unreacted. Peptide synthesis can generally be carried out according to the following process.</p><p> The carrier resin Fmoc-NH is placed in the reaction vessel of the automatic synthesizer. Add DMF to this and stir for 3 minutes. The solution is then drained. The 30% piperidine-DMF solution is then added, the mixture is stirred for 4 minutes, the solution is drained and this operation is repeated.</p><p> The carrier resin is then washed with DMF for 1 minute, the solution is drained and this operation is repeated 5 times. Protected amino acid residues are then added sequentially and a partial deprotection step is intervened to ensure that the residues are added at the correct position and in the correct sequence.</p><p> The peptide can then be cleaved from the resin with the side chain protecting groups removed. The crude product is then dissolved in aqueous acetic acid and then passed through a cartridge filled with reverse phase silica gel to allow the peptides to adhere. Washing with 0.1% TFA and 15% acetonitrile aqueous solution and eluting with 0.1% TFA and 25% acetonitrile aqueous solution gives a fraction containing the peptide.</p><p> The peptide is generally synthesized with a terminal cysteine residue, which allows the peptide to be then conjugated to KLH, resulting in the immunogenicity of the peptide. For example, prepare a 10 mg / ml KLH solution by dissolving KLH in PBS, add 1/10 volume of 25 mg / ml MBS (N- (m-maleimidebenzoyloxy) succinimide), and then stir. Let it react for 30 minutes. Next, 2.5m g KLH-MB (which is obtained by removing free MBS using a Sephadex G-25 column pre-equilibrium in PBS) in 0.1 M sodium phosphate buffer (pH 7.0). Mix with 1 mg of dissolved peptide. This is reacted at room temperature for 3 hours with stirring. After the reaction, the product can be dialyzed against PBS and used as an immunogen.</p>

<p> Example 6. Maintaining antibody activity during acid / pepsin treatment To investigate the protective effect of bovine primal milk as a protective agent for antibody activity in the gastric environment, simulated gastric juice was tested with a specific monoclonal antibody. The activity was compared in the presence and absence of bovine udder.</p><p> A virus-specific IgG2a monoclonal diluted 1:10 in phosphate buffered saline was used in this example. The simulated gastric juice was a 0.32% solution of porcine pepsin in 0.03 M NaCl and adjusted to pH 1.2 with HCl. Degreased lyophilized cow dairy milk was obtained from unimmunized cows as a source and reconstituted at 300 mg / mL in water.</p><p> An aliquot of the antibody diluted 1:10 in phosphate buffered saline was mixed with either equal volume of reconstituted primary milk or water. A mixture of dairy preparation and water was used as a control. The mixture was preheated for 5 minutes in a 37 ° C water bath prior to the antibody activity assay.</p><p> Treatment of the monoclonal antibody with simulated gastric juice resulted in a 6-fold reduction in antibody activity. In contrast, addition of primary milk to the antibody preparation provided protection of the antibody from the effects of pepsin and acids. In the presence of first milk, the protected antibody did not show reduced activity. Using this antibody model, bovine dairy milk was shown to provide protection for antibody activity in a simulated gastric environment.</p>

<p> Example 7. Use of antibody-containing milk product A patient presents with an unknown esophageal condition. Esophageal biopsy material is taken and EphB4 expression levels are examined and found to be higher than the surrounding normal esophageal tissue. It is concluded that the patient has Barrett's esophagus. Patients receive (orally) daily doses of milk products containing antibodies directed to EphB4. Continued administration of this milk product has a therapeutic effect and the patient's Barrett's esophageal symptoms are alleviated.</p>

<p> Example 8. Antibody production in chickens Once the latent target molecule and its epitope have been identified, prepare sufficient amounts of antigen to immunotreat up to 10 adult female chickens. The amount of peptide is known to those of skill in the art and can be easily optimized with minimal experimentation.</p><p> The diluted antigen mixture is injected into each chicken and repeated immunization is performed every 2 weeks for 6 months. One month after the first immune procedure, egg collection from chickens was started.</p>

<p> Example 9. Use of antibody-containing egg product Previously, antibodies or fractions in eggs laid by female birds hyperimmunized with specific bacterial antigens have been shown to survive and remain active in the intestinal tract. Thus, as in Example 5, if a patient exhibits Barrett's esophagus, the patient may be (orally) administered a daily dose of egg product containing an antibody directed to EphB4. The egg product is "Tamagozake" per day. It can be in the form of "egg flip)". Tamagozake can be made using spray-dried egg yolk as an ingredient. Continued administration of the egg product then has a therapeutic effect and alleviates the patient's Barrett's esophageal symptoms. Such protocols can also be used to treat and / or prevent cancer of the GI tract.</p><p> The present invention is not limited to the scope of the specific embodiments described herein. The embodiments are intended to be examples of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. In fact, various variations of the invention will be apparent to those skilled in the art from the above description and accompanying drawings in addition to those shown and described herein. Such modifications shall be included in the scope of the appended claims.</p>