JP2012518407A - Isolated monocyte population and related therapeutic applications - Google Patents

Abstract

Summary The present invention provides methods of using isolated monocyte populations to treat patients suffering from various ocular vascular diseases or ocular degenerative disorders. The present invention also provides a novel method for isolating a substantially pure monocyte population. The method includes extracting a blood sample or bone marrow sample from a patient, debulking red blood cells from the sample, and then separating residual red blood cells and other cell types in the sample from monocytes. Without any selective or labeling agent, red blood cells or other cell types are separated from monocytes based on their size, particle size or density. Isolated monocytes can be further activated in vitro or ex vivo prior to administration to a patient. Isolated cell populations containing substantially pure CD14 + / CD33 + monocytes are also provided in the present invention.

Description

US Government Assistance Statement This invention was subsidized in part by the US Government with grant numbers EY11254, EY14174 and EY0175440 from the National Institutes of Health. Therefore, the US government has certain rights in the invention.
Cross-reference of related applications

No. 61 / 208,173 (filing date: February 20, 2009) and 61 / 283,244 (filing date: November 30, 2009).
Claim priority interests based on. The entire information disclosure of the priority application is incorporated herein by reference in its entirety for all purposes.
Background of the Invention

  Ocular vascular diseases such as age-related macular degeneration (ARMD) and diabetic retinopathy (DR) are caused by abnormal choroidal neovascularization or retinal neovascularization. These are the main causes of blindness in developed countries. Because the retina consists of distinct layers of neurons, glial cells and vascular components, relatively minor disorders such as those seen in vascular proliferation or edema can lead to a significant loss of visual function. Hereditary retinal degeneration such as retinitis pigmentosa (RP) is also associated with vascular abnormalities such as arteriole stenosis and vascular atrophy. Hereditary retinal degeneration develops in 1 in 3500 people and develops central night vision of progressive night blindness, visual field loss, optic nerve atrophy, arteriolar attenuation, and often progresses to complete blindness. Characterized by. Although significant progress has been made in identifying factors that promote and inhibit angiogenesis, there are still no effective therapies to slow or reverse the progression of these retinal degenerative diseases.

  There is a need in the art for better methods for treating and preventing various ocular vascular diseases. The present invention is directed to this and other needs.

In one aspect, the present invention provides an isolated cell population containing substantially pure monocytes that express CD33 and CD14 antigens. Some of these isolated cell populations are isolated from mammalian peripheral blood samples, umbilical cord blood samples or bone marrow samples. Isolated cell populations also include human cells or mouse cells. In some of the isolated cell populations, at least 70%, 80% or 90% of the cells express the surface markers CD14 and CD33. Some of the isolated cell populations do not contain cells that express CD34. Some of the isolated cell populations are substantially free of ALDH ^br cells. The isolated cell population can be further activated in vitro or ex vivo. This is achieved by any monocyte activating compound such as, for example, LPS, MPLA, or MCP-1.

In another aspect, the present invention provides a method for treating ocular vascular disorders. The method includes administering to a patient suffering from an ocular vascular disorder a sufficient amount of an isolated monocyte population to treat or ameliorate the ocular vascular disorder. Preferably, the monocyte population is isolated from a blood sample or bone marrow sample from the patient. In some preferred embodiments, the patient to be treated by the method is a human. In some of the methods, the monocyte population comprises substantially pure CD14 ⁺ / CD33 ⁺ cells. For example, at least 80% of the cells in the isolated monocyte population are CD14 ⁺ / CD33 ⁺ . Monocyte populations isolated in some methods are activated in vitro or ex vivo prior to administration to a patient. In these embodiments, any compound known to activate monocytes can be used. For example, isolated monocyte cells can be activated with LPS, MPLA, or MCP-1. In some methods, an untreated monocyte population (or an in vitro or ex vivo activated monocyte population) is co-administered to a patient with such a monocyte activating compound.

  Many types of ocular vascular disorders can be treated by the methods of the present invention. Examples include venous stasis retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlusion, retinal edema, macular degeneration, and retinitis pigmentosa. In some methods, the isolated monocyte population is administered to the patient by a local route, for example, intravitreal injection. In some other methods, the isolated monocyte population is administered to the patient by a systemic route such as, for example, intravenous injection.

  In a related aspect, the present invention provides other methods of treating or ameliorating ocular disease in a patient. These methods include (i) isolating a substantially pure monocyte population from a blood sample or bone marrow sample of a patient suffering from ocular vascular disease; and (ii) treating or ameliorating ocular vascular disease. It is necessary to administer the isolated monocyte population to the patient in an amount sufficient to make it happen. In addition, some of these methods require that the isolated monocyte population be activated ex vivo prior to administering the cells to the patient. Any compound known to activate monocytes can be used in these embodiments. For example, isolated monocyte cells can be activated by LPS, MPLA, or MCP-1. In some other embodiments, the isolated monocyte population with or without ex vivo activation is co-administered to the patient with the monocyte activating compound.

Typically, the monocyte population used in these methods contains substantially pure CD14 ⁺ / CD33 ⁺ cells. Preferably, at least 80% of the cells in the isolated monocyte population express the surface markers CD33 and CD14. In some methods, the monocyte population is (i) debulking red blood cells from the sample, and (ii) separating residual red blood cells and other cell types in the sample from monocytes based on their size, particle size, or density. To be isolated. In some of these methods, residual red blood cells and other cell types are separated from monocytes by density centrifugation or fluorescence excited cell sorting (FACS). Ocular diseases or disorders suitable for treatment by these methods include venous stasis retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlusion, macular degeneration, and retinitis pigmentosa Is included.

  In another aspect, the present invention provides a method for isolating a substantially pure monocyte population. The method includes (i) providing a blood sample or bone marrow sample from a patient; (ii) debulking red blood cells from the sample; and (iii) residual red blood cells and other cell types (platelets and granules) in the sample Separating spheres) from monocytes. In some of the methods, residual red blood cells and other cell types are separated from monocytes based on their size, particle size or density. In some of the methods, residual red blood cells and other cell types are separated from monocytes by density centrifugation or fluorescence-excited cell sorting (FACS). In these methods, red blood cells can be debulked by Hespan differential centrifugation or Ficoll density gradient centrifugation. These methods can also include an analysis step for expression of the surface markers CD14 and CD33 of the isolated cell population.

  A better understanding of the nature and advantages of the present invention will be obtained by reference to the remaining portions of the specification and claims.

Figures 1A-1B show the properties and therapeutic activity of the isolated monocyte population. (A) Flow cytometry plot showing a population of monocytes different from lymphocytes (boxed area). No label was used to distinguish between these populations; and (B) human peripheral blood (HuPB) monocytes isolated by the described method were angiogenic tufted branches compared to control vehicle injections. Data obtained from an oxygen-induced retinopathy mouse model demonstrating significant reduction in both tuft area (black bars) and vascular occlusion (white bars). These results were similar to mouse bone marrow-derived CD44hi cells used as positive controls. 2A-2B show the results of flow cytometric analysis of the fraction produced by density centrifugation. The data show that CD2 ⁺ / CD3 ⁺ lymphocytes in the sample are depleted (A) and CD14 ⁺ / CD33 ⁺ monocytes are increased (B). FIG. 3 shows the results of ALDH labeling of peripheral blood, showing that the ALDH ^br / SSC population is negligible. FIG. 4 shows the results of a flow cytometry analysis showing that the presence of CD34 ⁺ cells (upper right) is small in relation to target CD14 ⁺ monocytes (upper left) in the isolated cell population. FIG. 5 shows the analysis after selection of human peripheral blood monocytes or lymphocytes selected based on the above light scattering characteristics. The monocyte fraction is composed of ˜88% CD14 ⁺ cells, while the lymphocyte population is shown to be substantially free of CD14 ⁺ cells. Also shown is an analysis for CD11b and CD33 that demonstrates high expression of both these myeloid markers on the monocyte fraction and a small number of positive cells in the lymphocyte fraction. FIG. 6 shows the results of an in vitro cell migration assay that reveals a dose-dependent increase in monocyte (Mono) migration in response to MCP-1. Lymphocytes (Lympho) did not respond to MCP-1. Mouse CD44hi bone marrow cells (CD44Hi) containing monocytes also responded to MCP-1. FIG. 7 shows an in vitro differential adhesion assay that demonstrates the ability to increase the number of monocytes that adhere to untreated cell culture plastic. A significant number of lymphocytes failed to adhere to the substance. FIG. 8 shows an image from a retinal whole tissue specimen showing the presence of GFP-expressing cells in the retina within 5 days after intracardiac injection. Injuries were created in the retina by exposure to hyperoxia. FIG. 9 shows a cytometric bead array (CBA) analysis of secreted cytokines derived from LPS-treated monocyte-enhanced F5 cells (ActF5). The data revealed increased secretion of IL-1beta, IL-6, IL-8 and TNF after LPC stimulation. For each cytokine, an ED50 approximation is given as a measure for the amount of protein present in the medium and biological activity. The unit is pg / ml. FIG. 10 shows cytometric bead array data revealing increased cytokine secretion after 1 hour or 4 hour incubation with LPS, MPLA or mouse MCP-1. Two concentrations of LPS and MPLA are shown. Values represent the ratio of treated (activated) cells to untreated (control) cells. FIG. 11 shows cytometric bead array data after 4 and 19 hour stimulation with LPS, mouse MCP-1, human MCP-1 and MPLA at different concentrations. At 19 hours, in addition to LPS and MPLA, mouse and human MCP-1 are shown to stimulate IL-8 and IL-6 secretion despite low levels. FIG. 12 shows that active monocyte-enhanced fraction 5 (F5) from both healthy and diabetic donors promotes vascular repair in the OIR mouse model more efficiently than non-active F5 or other fractions. . The data shown shows the percentage of vaso-occlusive retinas less than 10,000 square microns within the treatment group. Detailed Description of the Invention Summary of the Invention

The present invention relates to an isolated substantially pure monocyte population useful for treating or ameliorating ocular vascular disease or ocular degenerative disorders. As detailed in the examples below, the monocyte population isolated according to the present invention contains substantially pure CD14 ⁺ / CD33 ⁺ monocytes. The isolated monocyte population has a blood vessel repair promoting effect as tested in an ocular disease model. This monocyte population is not labeled with AldeFluor Bright and is different in nature from other known hematopoietic cells used clinically, as shown by its therapeutic activity independent of CD34 ⁺ cells. Furthermore, some of the isolated cell populations are also characterized by being CD34− and / or containing very low levels of aldehyde dehydrogenase expressing cells (ALDHbr cells). In addition, the inventors have found that some of the isolated monocyte populations enhance the ability to promote vascular repair by ex vivo activation. Finally, like cells isolated from healthy donors, monocytes isolated from donors suffering from retinal vascular disorders are also activated ex vivo for vascular repair in a mouse model of venous congestive retinopathy It has been found to promote. These findings further support that therapeutically active monocyte populations can be used in autografts to treat retinal vascular disorders.

  The inventors have also developed a new method for isolating monocyte populations for the treatment of neovascular eye diseases such as macular degeneration and diabetic retinopathy. When using biological samples such as bone marrow, peripheral blood or umbilical cord blood, this method requires the use of a selection reagent that specifically recognizes monocyte surface antigens such as antibodies, utilizing the physical properties of the target cell population. do not do. Cell populations isolated by this method are more desirable for therapeutic applications because they do not contain heterogeneous material bound to the surface, such as antibodies. A series of in vitro assays were performed to determine the purity and activity of these monocyte preparations. Furthermore, monocyte populations isolated using the methods disclosed herein have been found to have desirable therapeutic activity in a venous congestive retinopathy model.

  In accordance with these discoveries, the present invention provides isolated or substantially purified monocyte populations that are therapeutically effective. The present invention also provides a novel method for isolating the monocyte population. The present invention further provides a method of treating or ameliorating a disease or disorder associated with or mediated by abnormal ocular neovascularization. In addition, methods for producing highly active monocyte cells by in vitro or ex vivo activation with compounds capable of activating monocytes (eg, agonist compounds of CD14 or TLR4), and similar methods Also provided are methods for identifying novel compounds that can activate sphere cells. The present invention also encompasses a therapeutic method utilizing a combination of an isolated monocyte population and a compound capable of activating and restoring cells (eg, MCP-1). In these methods, cells can be activated based on administration to a patient and mediate sustained effects with additional replacement cells.

Highly activated cells and novel activated compounds are useful for the treatment of various ocular diseases. Examples of such diseases include diabetic retinopathy, diabetic macular edema, retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, retinal hemangiomatous proliferation, macular telangiectasia, venous congestion Retinopathy, iris neovascularization, intraocular neovascularization, corneal neovascularization, retinal neovascularization, choroidal neovascularization, and retinal degeneration are included. Patients suitable for treatment by the methods of the present invention include those who develop or are at risk of developing any of these diseases. The following sections provide detailed guidance for implementing the method of the present invention.
II. Definitions Unless otherwise specified, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of a number of terms used in the present invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1 ^st ed., 1992). ^{; (. Ed) Illustrated Dictionary of} Immunology, Cruse, CRC Pr Llc (2 nd ed, 2002.); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revise ed., 2000); Encyclopedia of Chemistry, Kumar (Ed.), Anmol Publicactivities Pvt. Ltd .. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. ^{(. Eds), John Wiley &} Sons (3 rd ed, 2002.); (. Ed) Dictionary of Chemistry, Hunt, Routledge (1 st ed, 1999.); (. Ed) Dictionary of Pharmaceutical Medicine, Nahler, Springer -Verlag Telos (1994); Dictionary of Organic Chemistry, Kumar and Andand (Eds.), Anmol Publications Pvt. Ltd .. (2002); (. Eds) and A Dictionary of Biology (Oxford Paperback Reference ), Martin and Hine, Oxford University Press (. 4 th ed, 2000). Furthermore, the following definitions are prepared for the convenience of the reader in practicing the present invention.

  Unless otherwise specified, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

  Hematopoietic stem cells are stem cells that can differentiate into various types of blood cells, such as B cells, T cells, granulocytes, platelets, and erythrocytes. Lineage surface antigens (surface markers) are CD2, CD3, CD11, CD11a, Mac-1 (CD11b: CD18), CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, CD45RA, mouse Ly-6G, Of cell surface proteins that are markers of mature blood cell lineages including mouse TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR), and CD235a (glycophorin A) It is a group. Hematopoietic stem cells that do not express significant levels of these antigens are commonly referred to as lineage negative (Lin_). Human hematopoietic stem cells generally express other surface antigens such as CD31, CD34, CD117 (c-kit) and / or CD133. Mouse hematopoietic stem cells generally express other surface antigens, CD34, CD117 (c-kit), Thy-1, and / or Sca-1.

Cells that circulate in the bloodstream are generally classified into three types: white blood cells, red blood cells, and platelets. Leukocytes include granulocytes (polynuclear leukocytes) and agranular leukocytes (mononuclear leukocytes). Granulocytes are white blood cells characterized by the presence of a group of granules in the cytoplasm that are stained differently when observed under an optical microscope. There are three types of granulocytes: neutrophils, basophils, and eosinophils. Agranular leukocytes (mononuclear leukocytes) are white blood cells characterized by the apparent absence of granules in the cytoplasm. Although the name implies the absence of granules, these cells contain non-specific azurophilic granules, lysosomes. Agranular leukocytes include lymphocytes, monocytes, and macrophages.

  Monocytes are produced by the bone marrow from hematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about 1 to 3 days, and then typically travel throughout the body into the tissue. Monocytes make up 3 to 8 percent of the white blood cells in the blood. In tissues, monocytes mature into different types of macrophages at different anatomical sites. Monocytes have two main functions in the immune system: (1) in normal conditions, recruiting resident macrophages and dendritic cells, and (2) in response to inflammatory signals, The sphere moves quickly to the site of infection in the tissue (about 8-12 hours) and divides / differentiates into macrophages to elicit an immune response. Monocytes are usually identified by their large two-slice nuclei in the stained smear.

  Ocular neovascularization or ocular vasculature disorder is a pathological condition characterized by altered or uncontrolled growth and invasion of new blood vessels into the structure of ocular tissues such as the retina or cornea. Examples of ocular neovascular diseases include venous congestive retinopathy, iris neovascularization, intraocular neovascularization, age-related macular degeneration, corneal neovascularization, retinal neovascularization, choroidal neovascularization, diabetic retinal ischemia, Retinal degeneration and diabetic retinopathy are included.

  Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, upper ring keratoconjunctivitis, pterygium , Sjogren's syndrome, erythematous acne, friculosis, syphilis, mycobacterial infection, steatosis, chemical burn, bacterial ulcer, fungal ulcer, herpes simplex, herpes zoster infection, protozoal infection, Kaposi's sarcoma, Mohren Includes ulcers, perithenic degeneration, peripheral keratolysis, rheumatoid arthritis, systemic erythema, polyarteritis, trauma, Wegener sarcoidosis, scleritis, Stevens-Johnson syndrome, radial keratotomy, and corneal graft rejection.

  Other diseases associated with retinal / choroidal neovascularization include, but are not limited to, diabetic retinopathy, macular degeneration, sickle cell anemia, sarcoid, syphilis, elastic fibrotic pseudoxanthoma, Paget's disease Venous occlusion, arterial occlusion, carotid occlusive disease, chronic uveitis / vitreitis, mycobacterial infection, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, retinitis pigmentosa, retinal edema (including; Macular edema), Eales disease, Behcet's disease, retinitis or choroiditis-induced infection, putative ocular histoplasmosis, best disease, myopia, papillary cavity, Stargardt disease, flatitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasma Complications, trauma, and complications after laser surgery. Other diseases include, but are not limited to, diseases associated with skin flushing (angular neovascularization) and abnormal growth of fibrovascular or fibrous tissue, including all forms of proliferative vitreoretinopathy Disease included.

  Retinopathy of prematurity (ROP) is an eye disease that affects infants born prematurely. It is thought to be caused by the erratic proliferation of retinal blood vessels and can result in scars and retinal detachment. ROP may be mild and recover spontaneously, but in severe cases it can be blind. All premature infants have a corresponding risk of ROP and significantly lower birth weight can be an additional risk factor. Both oxygen toxicity and relative hypoxia can contribute to the development of ROP.

  Macular degeneration is a medical condition that is primarily seen in the elderly, with the center of the inner back of the eyeball, known as the macular region of their retina, exhibiting reduced wall thickness, atrophy, and sometimes bleeding. This can cause loss of central vision, which inevitably makes it impossible to see details, read or recognize the face. According to the American Academy of Ophthalmology, macular degeneration is still the leading cause of central vision loss (blindness) in the United States today for over 50 years. Macular dystrophy that affects young individuals is often referred to as macular degeneration, but the term generally refers to age-related macular degeneration (AMD or ARMD).

  Age-related macular degeneration begins with a characteristic yellow deposit in the macula called drusen (the central area of the retina that provides a fine central vision called the fossa) between the retinal pigment epithelium and the basal choroid . Most people who suffer from these initial changes (called age-related macular degeneration) have good vision. People with drusen may develop advanced AMD. The risk is quite high when drusen is large, numerous, and involves the submacular pigment cell layer. Large and soft drusen is associated with increased cholesterol deposits and can be improved with cholesterol lowering agents or Rheo Procedures.

  There are two types of advanced AMD that cause severe visual loss, dry and wet. Central AMD, a dry form of advanced AMD, is caused by atrophy to the subretinal retinal pigment epithelium and causes visual loss due to loss of photoreceptors (rods and cones) in the central part of the eyeball. No treatment is useful for this condition, but supplementing vitamins with high doses of antioxidants, lutein and zeaxanthin slows the progression of dry macular degeneration and reduces vision in some patients Improved by the National Eye Institute and other laboratories.

  Retinitis pigmentosa (RP) is a group of hereditary eye diseases. In the progression of RP symptoms, night blindness usually develops years or decades prior to the tunnel vision. Many people who suffer from RP are not legally blind until their 40s or 50s and maintain some vision throughout their lives. Other people are completely blinded by RP as early as in some cases. The progression of RP varies in individual cases. RP is a type of hereditary retinal dystrophy, a group of genetic disorders in which abnormalities in photoreceptor cells (rods and cones) or retinal pigment epithelium (RPE) in the retina lead to progressive visual loss. Patients initially experience dark adaptation deficits or night blindness (night blindness), followed by a decrease in peripheral vision (known as a tunnel vision), and sometimes loss of central vision at the end of the disease course.

  Macular edema occurs when fluid and protein deposits deposit on or below the macula of the eyeball, the yellow central region of the retina, causing macular thickening and swelling. Since the macula is approximately in the middle of the retina behind the eyeball, its swelling probably distorts the person's central vision. This region contains closely packed cones that provide a clear and clear central vision to allow humans to see forms, colors, and details that are directly in the viewing direction. Cystic macular edema is macular edema that includes cyst formation.

  The terms “subject” and “patient” are used interchangeably, mammals such as human patients and non-human primates, as well as laboratory animals such as rabbits, rats, and mice, and many animals. Point to. The animals include all vertebrates such as mammals and non-mammals such as dogs, cats, sheep, cows, pigs, rabbits, chickens and the like. A preferred patient for practicing the treatment methods of the invention is a human. Patients in need of treatment include patients with a history of ocular vascular disease or disorder as well as patients who are susceptible to developing the disorder.

  When referring to an isolated cell population, the term “substantially pure” or “substantial purity” means that the percentage of a given cell (target cell) in the population is in the natural environment (eg, patient tissue or blood). Means significantly higher than found in the flow). Typically, the percentage of target cells (eg, monocytes) in a substantially pure cell population is at least about 50%, preferably at least about 60%, 70%, 75%, of all cells in the cell population, And more preferably at least about 80%, 85% 90% or 95%.

As used herein, “treating” or “ameliorating” includes (i) preventing (eg, preventing) the occurrence of a pathological condition (eg, macular degeneration); (ii) Inhibiting or preventing the development of a pathological condition (eg, macular degeneration); and (iii) alleviating symptoms associated with the pathological condition (eg, macular degeneration). Thus, “treatment” includes isolated cell populations of the invention and / or for preventing or delaying the onset of symptoms, complications, or biochemical signs of the ocular diseases described herein. Administration of other therapeutic compositions or agents is included, thereby alleviating or ameliorating symptoms or preventing or inhibiting further progression of the disease, condition, or disorder. “Treatment” further allows the patient to tolerate any indication of success in the treatment or amelioration or prevention of the eye diseases, symptoms, or disorders described herein, ie, remission; remission; reduction of symptoms or pathology. Refers to signs that include any objective or subjective factors such as: slowing down the rate of degeneration or decay; or lessening the decay at the end of denaturation. Detailed procedures for the treatment or remission of ocular disorders or symptoms thereof can be based on objective or subjective factors including test results by a physician.
III. Methods for isolating monocyte cell populations

The present invention provides methods for isolating monocyte populations useful for treating the various ocular vascular disorders described herein. As illustrated in the examples below, a monocyte population can be isolated from a suitable biological sample taken from a mammalian patient, such as peripheral blood or bone marrow. The methods of the invention allow for the isolation of a substantially pure monocyte population from bone marrow or blood samples (eg, purity of at least 50%, 75%, or 85%). The blood sample can be any sample containing a large amount of white blood cells or mononuclear leukocytes derived from whole blood. For example, it can be whole blood or a leukocyte separation product derived from whole blood. Leukocyte separation is an experimental procedure for separating white blood cells from a blood sample. Preferably, the monocytes present in the isolated cell population are CD14 ⁺ / CD33 ⁺ . CD33 is a transmembrane receptor expressed in monosite / myeloid cells. CD14 is a membrane-bound glycosylphosphatidylinositol-linked protein that is expressed on the surface of cells, particularly macrophages. Bone marrow, peripheral blood, and umbilical cord blood each contain a subpopulation of monocytes that express the CD14 antigen and CD33. Accordingly, these biological samples are preferred for isolating monocyte populations enriched in CD14 ⁺ and CD33 ⁺ cells by the methods described herein. In some embodiments, the isolated cell population is also characterized by being CD34 ⁻ and / or not expressing or expressing low levels of aldehyde dehydrogenase (ALDH). Preferably, the monocyte population is isolated from human bone marrow, human peripheral blood, human umbilical cord blood or other related blood sample.

  Typically, the method requires an initial removal (“debulking”) of most red blood cells (RBCs) from the sample. This stage involves the separation of other blood cells (eg, platelets, granulocytes and lymphocytes) and any remaining red blood cells from monocytes. Unlike methods known in the art, labeling agents (eg, antibodies) that recognize cell surface markers of various cell types are not used in the methods of the invention. Instead, the present invention separates monocytes from other blood cell types, particularly other mononuclear cells (eg, lymphocytes) based solely on physical properties such as size, granularity and density. In some embodiments, the monocyte population of the invention is isolated from a suitable sample such as bone marrow or peripheral blood by a method based on fluorescence-excited cell sorting (FACS). As detailed in the examples below, RBCs present in a biological sample (eg, peripheral blood) from a mammalian patient are removed at the beginning of the isolation procedure. This can be achieved by dissolution of RBCs by standard procedures well known in the art, such as dissolution methods based on ammonium chloride. See, for example, Tiirikainen, Cytometry 20: 341-8, 1995; and Simon et al. , Immunol. Commun. 12: 301-14, 1983. Alternatively, RBCs can be sedimented and mononuclear cells separated by ficoll centrifugation. Red blood cell separation procedures by Ficoll density gradient centrifugation are disclosed in the art. For example, Tripodi et al. , Transplantation. 11: 487-8, 1971; Vissers et al. , J. et al. Immunol. Methods. 110: 203-7, 1988; and Boyum et al. , Scand. J. et al. Immunol. 34: 697-712, 1991. Another suitable method for RBC debulking is by differential centrifugation using the ability of Hespan (Dupont, Drieich, Germany) to induce erythrocyte aggregation. For example, Nagler et al. , Exp. Hematol. 22: 1134-40, 1994; and Pick et al. , Br. J. et al. Haematol. 103: 639-50, 1998. Further methods that can be used to debulk RBCs include the use of blood cell filters. Such a blood cell filter, for example, a leukocyte removal filter manufactured by Pall Biomedical Products Company (East Hills, New York) can be easily obtained from a commercial supplier.

  After removal of the RBCs, the remaining cells in the sample are suspended in a suitable buffer that is compatible with subsequent FACS isolation steps. For example, the cells can be suspended in DPBS / 0.5% BSA / 2 mM EDTA. Flow cytometry is a method for counting, measuring and sorting fine particles suspended in a flow of sheath fluid. It allows simultaneous multivariable analysis of the physical and / or chemical properties of a single cell flowing through optical and / or electronic detection devices. Typically, a single wavelength beam (usually a laser beam) is directed to a hydrodynamically concentrated flow of the sheath fluid. Some detectors aim at the point where the flow passes through the beam. One detector is collinear with the beam (forward scatter or FSC) and some are perpendicular to it (side scatter (SSC) and one or more fluorescence detectors). Individual suspended particles that pass through the light beam scatter the light beam in some way, and the fluorescent chemicals present in or attached to the particle are excited into a lower frequency emission beam than the light source. This combination of scattered light and fluorescence is received by the detector and then analyzed for luminance variations at each detector (one for each fluorescence emission peak), and then the physical and chemical properties of each individual particle. Various types of information about the structure can be derived. FSC is related to cell volume, and SSC depends on the internal complexity of the particle (eg, nuclear morphology, cytoplasmic granule quantity and type, or membrane roughness). Some commercially available flow cytometers eliminate the need for fluorescence and use only light scattering for measurements. Other flow cytometers form an image of each cell's fluorescence, scattered light, and transmitted light.

  Modern flow cytometers can quickly analyze thousands of particles per second and can actively separate and isolate particles with certain properties. Instead of creating an image of the cell, the flow cytometer is similar to a microscope, except that the set parameters are automatically quantified quickly and in large quantities. A flow cytometer consists of a flow cell-sheath fluid flow, a light source (eg, a laser), a detector and an analog-to-digital conversion (ADC) system, a signal amplification system, and a signal analysis computer that generates a fluorescence signal similar to FSC or SSC. It has 5 main components. The data generated by the flow cytometer can be dot plotted to create a histogram, or can be dot plotted in two dimensions or even dot plotted in three dimensions. These plotted regions can be separated sequentially based on fluorescence intensity by creating a series of sub-populations named “gates”. There are special gating protocols, especially for diagnostic and clinical purposes related to hematology. Plots are often made on a logarithmic scale. In order for the emission spectra of different fluorochromes to overlap, the signal at the detector must be compensated electronically and computationally.

  Fluorescence excited cell sorting (FACS) is a special type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells one cell at a time into two or more containers based on the specific light scattering and fluorescence characteristics of each cell. It is a useful scientific instrument because it provides rapid, objective and quantitative recording of fluorescent signals from individual cells, as well as physical sorting of cells of special interest. The cell suspension is placed in the center of a rapidly flowing narrow sheath fluid stream. The flow is adjusted so that there is a large spacing between cells in proportion to their diameter. An oscillating mechanism turns the flow of cells into individual droplets. The system is adjusted to reduce the possibility of two or more cells entering one droplet. Immediately before the flow changes to a droplet state, the flow passes through the observation point of fluorescence measurement, where the fluorescence characteristics of each target cell are measured. The charging ring is installed at a point where the flow changes to a droplet state. The charge is placed on the ring based on the previous fluorescence intensity measurement, and the opposite charge is trapped in the droplet as the droplet changes from flow. The charged droplets then fall through an electrostatic deflector that delivers the droplets to the container based on the charge. In some systems, the charge is directly applied to the flow and the broken droplet retains the same charge as the flow. The flow is then returned to neutral after the droplet is cut off.

  As an example of the present invention, FACS can be performed by a BD FACSAria Cell-Sorting System (BD Biosciences, San Jose, Calif.) Using a series of gates. Do not use any antibody or other selective agent in the preparative. Dead cells and necrotic tissue pieces can be initially gated out by defining a region containing only live leukocytes. Thereafter, the two-cell conjugate or aggregated cell has a forward scattering width (FSC-W) for the forward scattering region (FSC-A) and a side scattering width (SSC-W) for the side scattering region (SSC-A). They can be removed by the second and third gates, respectively. Procedures are standard protocols well known in the art, such as Flow cytometry-A practical approach, Ormerod (ed), Oxford University Press, Oxford, UK (3rdred., 2000); and Handbook Forward. al. (Eds.), Wiley-Liss, New York (1993).

  In some other embodiments, the monocyte population of the present invention is isolated using a separation scheme based on the Elutra® cell separation system (Gambro BCT Inc, Lakewood, Colorado). Elutra® is a semi-automatic centrifugation-based device that uses a continuous counter-flow elicitation method that separates cells into multiple fractions based on size and density. Prior to separation with Elutra®, a biological sample taken from a mammalian patient (eg, peripheral blood from a human patient) is first removed by, for example, centrifugal precipitation with HESpan to remove large amounts of RBCs. Can be processed. The nucleated cell fraction can then be collected, for example, with a plasma expressor, prior to processing with the Elutra® device. As detailed in the examples below, fractionation with the Elutra® device allows separation of monocytes from platelets, residual RBCs, lymphocytes and granulocytes. The fractionated cells can be further analyzed for cell number, viability and purity.

In some embodiments, the present invention provides a method of producing highly active cells for therapeutic applications. In these embodiments, the monocyte population isolated according to the present disclosure is further activated in vivo or ex vivo prior to administration to a patient suffering from an ocular vasculature disorder. Typically, cells are treated with a compound capable of activating monocytes. Detailed procedures for activating the isolated monocyte population are described below.
IV. Characteristics and activity of isolated monocyte populations.

Monocyte populations isolated from biological samples such as whole blood or bone marrow can be tested for their therapeutic activity as well as their immunological or biological properties. As detailed in the examples, the purity and activity of the isolated monocyte population can be assessed by several assays. For example, to analyze the expression of surface markers, some methods of the invention can further comprise assessing the expression of CD14 and CD33 by the isolated monocyte population. Isolated cell surface marker expression can be tested with anti-CD14 and anti-CD33 monoclonal antibodies using flow cytometry. As illustrated in the Examples, the cell population isolated according to the present invention contains substantially purified CD14 ⁺ / CD33 ⁺ monocytes. For example, an isolated cell population can have at least 50%, 60%, 75%, 80%, 85%, 90% or 95% CD14 and CD33 expressing cells.

  In addition to being substantially pure, the isolated cell population has a functional efficacy to treat or ameliorate symptoms associated with ocular vasculature disorders. For example, a cell population isolated as disclosed herein can promote vascular repair in mouse oxygen-induced retinopathy. Venous congestive retinopathy model mice and their use in evaluating the therapeutic effect of isolated cell populations on ocular neovascular disorders have been disclosed in the art. For example, Ritter et al. , J. et al. Clin. Invest. 116: 3266-76, 2006; and Ritter et al. , Invest. Ophthalmol. Vis. Sci. 46: 3021-6, 2005.

  The function and biochemical activity of the isolated cells can be analyzed by measuring cell chemotaxis using, for example, monocyte chemotactic proteins such as MCP-1. Such activity measurements also provide readout information regarding the relative purity of the preparation and indicators of viability and function of the isolated cells. Methods for testing the purity and viability of isolated monocytes further include assays based on specific adhesion of monocytes to the cell culture base relative to other mononuclear cells. As is evident in the examples, the cells produced by the isolation method of the present invention were found to be predominantly monocytes as evidenced by their ability to adhere under the aforementioned assay conditions.

Some of the isolated monocyte populations of the present invention are also CD34 ⁻ . In addition to being CD14 ⁺ and CD33 ⁺ , the CD34 ⁻ monocyte population of the invention has no or very low levels of CD34 ⁺ cells (eg, about 5%, 4%, 3%, 2%, 1%,. Monocyte population containing only less than 5%, 0.25%, 0.1%, 0.05% or 0.01%). The presence of CD34 ⁺ cells in a cell population can be readily identified and quantified using methods well known in the art or disclosed herein. CD34 of the present invention ^- monocyte population are more suitable for use in several therapeutic applications of the present invention. It is known that CD34 ⁺ stem cells have the potential to differentiate into undesired cell types and may have the ability to proliferate. Such characteristics of CD34 ⁺ cells may be undesirable in the practice of the therapeutic methods disclosed herein. It was found that injection of undifferentiated stem cell populations such as CD34 ⁺ stem cells into mouse eyes resulted in poor results (Example 3). Thus, some monocytes population of the ^invention, in addition to being CD14 + / CD33 ^+, also characterized by the presence of CD34 ⁺ cells lacking or very low amount of CD34 ⁺ cells. As illustrated in the examples below, small amounts of CD34 ⁺ cells that may be present in the initial cell population can be further removed from the final isolated monocyte population. Importantly, removal of CD34 ⁺ cells as disclosed herein does not result in any change in the therapeutic effect of the monocyte population.

In some other embodiments, the monocyte population of the invention is also characterized by having no or substantially no aldehyde dehydrogenase high expressing cells (ALDH ^br cells). ALDH ^br cells are well known in the art. It has progenitor cell activity and has been suggested to be useful for cell therapy applications (Gentry et al., Cytother. 9: 259-274, 2007). The presence of ALDH ^br cells in a cell population can be determined from the examples herein and the art, for example, Ruso et al. , Biochem. Pharmacol. 37: 1639-1642, 1988; and Storms et al. , Blood 106: 95-102, 2005, and can typically be sorted and quantified by fluorescence-excited cell sorting (FACS). As shown in FIG. 3, some isolated monocyte populations of the invention contain very small amounts (about 0.04%) of ALDH ^br cells. Accordingly, some preferred embodiments of the invention provide an isolated or purified monocyte population that is substantially free of ALDH ^br cells. These monocyte cell populations should contain less than about 5%, 2%, or 1% ALDH ^br cells as measured by fluorescence-excited cell sorting. More preferably, the percentage of ALDH ^br cells in these cell populations should be less than 0.5%, less than 0.1%, or less than 0.05%. By being both CD34 ⁻ and / or low ALDH, these CD14 ⁺ / CD33 ⁺ monocyte populations of the present invention have been reported in the art (eg, Storms et al., Blood 106: 95-102, 2005) clearly distinguished from other blood cell or stem cell populations.

The cells derived from the monocyte population of the present invention are genetically engineered to express an anti-angiogenic substance for use in gene cell therapy or a therapeutically effective substance such as a neurotrophic substance for enhancing the neuronal rescue effect. can do. In these embodiments, the isolated monocyte cell population is transfected with a gene encoding a therapeutically effective substance. Suitable genes and methods for transfection of the monocyte population of the present invention into cells are described, for example, in US patent application Ser. No. 10 / 080,839. In some of these embodiments, the cells are transfected (eg, with a polynucleotide that operably encodes an angiogenesis-inhibiting peptide, such as TrpRS, or an anti-angiogenic (eg, anti-angiogenic) fragment thereof (eg, U.S. Patent Application No. 11 / 884,958). Engineered angiogenesis-inhibiting cells from the monocyte population regulate abnormal blood vessel growth in diseases associated with abnormal blood vessel development, such as ARMD, diabetic retinopathy, and certain retinal degeneration and similar diseases Useful for. In some other embodiments, the cells of the isolated monocyte cell population of the invention are transfected to express a gene encoding a neurotrophic substance. Neurotrophic substances expressed by the transfected gene are, for example, nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, retinal pigment epithelium-derived nerve It can be a trophic factor, insulin-like growth factor, glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and others. Monocyte cells transfected with such genes are useful to promote neuronal rescue in ocular diseases including retinal nerve degeneration such as glaucoma, retinal pigment degeneration, retinal nerve damage and the like. For example, Kirby et al. , Mol. Ther. 3: 241-8, 2001; Farrar et al. , EMBO J. et al. 21: 857-864, 2002; Fournier et al. , J. et al. Neurosci. Res. 47: 561-572, 1997; and McGee et al. , Mol. Ther. 4: 622-9, 2001.
V. Treatment of ocular vascular disease

  The present invention provides a method for treating or ameliorating vascular disorders and neurodegeneration in the retina of a mammal suffering from an ocular disease. According to the method, the isolated monocyte population or the above-described genetically engineered cells can be administered to a mammalian retina by either intravitreal injection or systemic administration. The cells are administered in an amount sufficient to ameliorate vascular degeneration and / or neural degeneration in the retina. Preferably, the isolated monocyte population is autologous to the mammal being treated. Preferably, isolated monocyte cells are administered in a physiologically acceptable medium such as phosphate buffered saline (PBS).

In some therapies, monocyte populations containing CD14 ⁺ / CD33 ⁺ cells that are substantially purified (eg, at least 75% or 80%) are initially collected from whole blood or bone marrow samples from patients in need of treatment. Isolated from The monocyte cell population is isolated using the method described above. The isolated CD14 ⁺ / CD33 ⁺ monocyte population is then administered to the patient in an amount sufficient to ameliorate vascular degeneration and / or neural degeneration in the retina. The cells are known from mammals suffering from ocular degenerative diseases or ocular vascular diseases, preferably at an early stage of ocular diseases, or susceptible to the development of ocular degenerative diseases (eg due to genetic predisposition). It can be isolated from healthy patients. In the latter case, the isolated monocyte population can be stored after isolation and then injected prophylactically during the early stages of ocular disease where symptoms appear later.

Without being bound by theory, the cells derived from the CD14 ⁺ / CD33 ⁺ monocyte population of the present invention selectively target astrocytes, enter the growing vasculature, and then enter vascular epithelial cells. It may exert its therapeutic effect by differentiating. The cells may promote neuronal rescue in the retina and promote upregulation of anti-apoptotic genes. When administered systemically or when injected intravitreally into the eyes of a mammalian patient (eg, a human or mouse) from which the cells were isolated, the cells are treated for retinal neovascularization and retinal vascular degenerative disease, and retinal blood vessels. Useful for repairing damage.

  Patients suitable for treatment with the methods of the invention can be neonates, young or fully mature adults. In some embodiments, the patient to be treated is a neonatal patient suffering from an ocular disease such as oxygen-induced retinopathy or retinopathy of prematurity. In some embodiments, the patient is human and the isolated monocyte population used is human cells, preferably autologous cells isolated from the patient to be treated. Patients suffering from various ocular vascular diseases or ocular degenerative disorders are suitable for treatment with the monocyte population of the present invention. These include retinal degenerative diseases, retinal vascular degenerative diseases, retinal edema (including macular edema) venous congestive retinopathy, bleeding, vascular leakage, choroidopathy, retinal damage and disruption or degeneration of the retinal vasculature Ocular diseases such as Specific examples of such diseases include age-related macular degeneration (ARMD), diabetic retinopathy (DR), putative ocular histoplasmosis (POHS), retinopathy of prematurity (ROP), sickle cell anemia, and Retinitis pigmentosa, as well as retinal damage are included. In addition, monocyte populations are used to create genetically identical cell lines, such as clones for the treatment or remission of retinal nerve degeneration as well as for use in regeneration or repair of retinal vasculature, for example. be able to. Furthermore, the monocyte population of the present invention is useful as a research tool for studying retinal vascular development and for delivering genes that select target cells such as astrocytes.

For therapeutic or prophylactic applications, the isolated monocyte population of the invention can be administered to patients by either local or systemic routes. In some embodiments, local administration of cells is desired to achieve the desired therapeutic effect. For example, a cell population can be administered to a patient by intraocular injection (intravitreal injection). This can be done according to standard procedures known in the art. For example, Ritter et al. , J. et al. Clin. Invest. 116: 3266-76, 2006; Russelakis-Carmeiro et al. , Neuropathol. Appl. Neurobio. 25: 196-206, 1999: and Wray et al. , Arch. Neurol. 33: 183-5, 1976. In some other treatment methods of the invention, systemic route administration of isolated monocyte populations is used. For example, the cells can be administered to the patient by intravenous injection routinely practiced in the art. In some other embodiments, non-human patients can also administer cells by intracardiac injection. This can be accomplished based on procedures routinely practiced in the art. For example, Iwasaki et al. , Jpn. J. et al. Cancer Res. 88: 861-6, 1997; Jespersen et al. , Eur. Heart J.H. 11: 269-74, 1990; and Martens, Resuscitation 27: 177, 1994. Other routes of administration may also be used in the practice of the present invention. For example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co. , 20 ^th ed. , 2000.

In general, the number of cells of the monocyte population injected into the eye should be sufficient to stop the disease state of the eye. For example, the amount of injected cells must be effective for repair of ocular retinal damage, stabilization of retinal neovascularization, maturation of retinal neovascularization, and prevention or repair of vascular leakage and vascular bleeding. For intravitreal injection, typically at least about 1 × 10 ⁴ , at least 1 × 10 ⁵ , or at least 1 × 10 ⁶ cells of an isolated monocyte population or transfected cells derived from a monocyte population are present in the ocular vasculature injury ( For example, it is injected into the eyeball of a patient suffering from a retinal degenerative disease. The number of cells injected will depend on the severity of retinal degeneration, the age of the patient and other factors that will be apparent to those skilled in the art of eye disease treatment. Cells from the monocyte population will be determined by the treating physician, but can be administered by single or repeated administration over a period of time. Cell number and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively small number of cells can be administered relatively infrequently over a long period of time. Some patients will likely continue to receive lifelong treatment. In therapeutic applications, a relatively large number of cells in a relatively short period of time until progression of disease symptoms is reduced or stopped, preferably until the patient exhibits partial or complete remission of symptoms of ocular vascular disease. May be required. Thereafter, it may be administered according to prophylactic management.
VI. Enhanced ex vivo activity of isolated monocyte populations

  In the various therapeutic applications described above, the isolated monocyte population or genetically engineered cells thereof can also be activated in vivo or ex vivo prior to administration to a patient in need of treatment. In these embodiments, ex vivo activated monocytes are administered to the retina of a patient suffering from ocular vascular injury resulting in enhanced therapeutic activity.

  Activation of the isolated monocyte population can be readily performed according to materials and methods routinely practiced in the art or exemplified in the examples below. Monocytes and macrophages are known to be activated by various agents such as LPS through CD14 and Toll-like receptors (Le-Barrillec et al., J. Leukoc. Biol. 68: 209-15). 2000; Mirlashari et al., Med. Sci. Monitor 9: BR316-24, 2003). As will be apparent in the examples below, the isolated monocyte population is divided into various agents such as lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA) and monocyte chemotactic protein 1 (MCP-1). Can be activated. It has also been demonstrated that activated cells produce better therapeutic results in animal oxygen-induced retinopathy compared to untreated cells. Thus, these agents can be used immediately for ex vivo activation of isolated monocyte populations. Many other monocyte activating compounds known in the art can also be used in the practice of the present invention. Examples of such compounds include immunomodulators (gamma interferon, lymphokyi, muramyl dipeptide, etc.), phorbol myristate acetate, concanavalin A, polymethyl methacrylate, and dietary fat. For example, Koff et al. , Science 224: 1007-1009, 1984; Chung et al. , J. et al. Leukoc. Biol. 44: 329-336, 1988; Horwitz et al. , J. et al. Exp. Med. 154: 1618-1635, 1981; Laing et al. , Acta Orthop. 79: 134-40, 2008; Bently et al. , Biochem. Soc. Trans. 35: 464-5, 2007.

  To activate monocytes, the isolated cells can be incubated with any of the compounds for a sufficient time at an appropriate concentration. The amount of compound used and the length of time for activation prior to administration of the cells can be determined empirically or according to the teachings of the art. Specific guidance for activating isolated monocyte populations with several compounds is also provided in the examples below. For example, when using LPS, the cells can be incubated with LPS at a concentration of about 1 ng / ml to 1000 ng / ml, preferably about 5 ng / ml to 200 ng / ml or about 20 ng / ml to 50 ng / ml. . The cells are typically treated with the activating compound prior to treatment, typically for at least 10 minutes, preferably for at least 1 hour. In some embodiments, the cells are treated with the compound for at least 2 hours, at least 4 hours, at least 10 hours, at least 24 hours or longer.

  Prior to administering the treated cells to a patient, the cells can also be tested in vitro to confirm their activity. This is typically done by qualitatively or quantitatively monitoring cytokine secretion by treated monocytes. As shown in the Examples, activated monocytes increased secretion of cytokines such as IL-1β, IL-8, IL-6 and TNF. As illustrated in the Examples, the cytokine secretion characteristics of monocytes can be easily evaluated by a commonly practiced method such as cytometric bead array (CBD) analysis. Elshal et al. , Methods, 38: 317-329, 2006; and Morgan et al. , Clin. Immunol. 110: 252-266, 2004.

  In addition to activating in vitro or ex vivo isolated monocyte populations prior to administering the cells to the patient, the present invention includes untreated monocyte populations and monocyte activating compounds as described herein (e.g., Several treatment methods are included in which MCP-1) is co-administered to the patient. In some related embodiments, a patient in need of treatment can be administered an in vitro or ex vivo activated monocyte population with a monocyte activating compound (eg, MCP-1) as described above. In these embodiments, the co-administered compound can activate the administered monocytes in vivo or can enhance the in vivo activity of the treated cells.

  In a related aspect, the present invention provides methods for identifying novel compounds capable of activating and stimulating monocyte therapeutic activity. Typically, these methods involve contacting a candidate compound with a monocyte or macrophage population (eg, a monocyte population described herein) and a monocyte parameter indicative of the activation state of the cell population. Need to monitor. The parameter to be monitored may be any biological, biochemical or morphological cellular characteristic. In some preferred embodiments, cells treated with a candidate agent are tested for secretion levels of one or more cytokines such as IL-6, IL-8 or TNF. Increased secretion of these one or more cytokines by treated cells compared to untreated cells indicates that the candidate compound is a novel monocyte activating compound.

Candidate compounds selected by this method can be derived from chemical species including small organic molecules, proteins, polypeptides, polysaccharides, polynucleotides and the like. In some preferred embodiments, the candidate compound is a small molecule organic agent (eg, an organic compound of less than about 500 Daltons or less than about 1,000 Daltons). Preferably, high-throughput analytical methods are adapted and used to screen combinatorial libraries of candidate compounds (eg, small organic molecule libraries). Such assays are described in the art, for example, Schultz (1998) Bioorg Med Chem Lett 8: 2409-2414; Weller (1997) Mol Divers. 3: 61-70; Fernandes (1998) Curr. Opin. Chem. Biol. 2: 597-603; and Sittampalam (1997) Curr. Opin. Chem. Biol. 1: 384-91. A large combinatorial library of candidate compounds is constructed by the code synthesis library (ESL) method described in WO 95/12608, WO 93/06121, WO 94/08051, WO 95/35503 and WO 95/30642. be able to. Other methods for synthesizing various libraries of compounds are described, for example, in Overman, Organic Reactions, Volumes 1-62, Wiley-Interscience (2003); Bloom et al. Fed Proc. 45: 2779-83, 1986; Ben-Menahem et al. , Recent Prog. Horm. Res. 54: 271-88, 1999; Schramm et al. , Annu. Rev. Biochem. 67: 693-720, 1998; Bolin et al. Biopolymers 37: 57-66, 1995; Karten et al. , Endocr. Rev. 7: 44-66, 1986; Ho et al. , Tactics of Organic Synthesis, Wiley-Interscience; (1994); and Scheit et al. , Nucleotide Analogs: Synthesis and Biological Function, John Wiley & Sons (1980).
Example

The following examples are provided to further illustrate the invention but are not intended to limit the scope of the invention. Other variations of the invention will be readily apparent to those skilled in the art and are encompassed by the appended claims.
Example 1 Isolation of Monocyte Population

  Peripheral blood or bone marrow can be used as a source material for the procedures described herein. For example, the inventors have selected peripheral blood as a cell source because of the relative abundance of monocytes relative to the bone marrow and the ease / safety of collection. For therapeutic applications, it is desirable to have cells that do not contain any binding compounds relevant to the selection. In light of this objective, the inventors planned and achieved a practical method of distinguishing monocytes from other mononuclear cells (eg, white blood cells) based solely on physical properties such as size, particle size and density. . The first method developed by the inventors is based on FACS to clearly separate monocytes from lymphocytes based on cell size and particle size without using antibodies. The results representing the monocyte population isolated by this method are shown in FIG. 1A. Red blood cells and granulocytes present in whole blood prior to FACS-based separation are removed during the Ficoll centrifugation step prior to sorting. This can be achieved by several means. For example, (1) ammonium chloride can be used to dissolve RBCs, (2) RBCs can be precipitated by centrifugation with Ficoll, and mononuclear cells can be isolated, and (3) Hespan RBCs can also be precipitated by use.

After debulking the RBCs, the cells are suspended in DPBS / 0.5% BSA / 2 mM EDTA with pretreatment for fluorescence-excited cell sorting (FACS). Sorting is performed by BD Biosciences ARIA using a series of gates without using antibodies or other selection agents. By defining an area containing only live leukocytes, dead cells and necrotic tissue pieces are first gated out. Next, the two-cell conjugate or aggregated cell is divided into a forward scattering width (FSC-W) for the forward scattering region (FSC-A) and a side scattering width (SSC-W) for the side scattering region (SSC-A). Are removed by the second and third gates. For only the other single leukocytes under consideration, gate in FSC-A vs. SSC-A mode to select cells found reproducibly in areas containing monocytes. Using the assay described in Example 2, the inventors found that the cell population collected by this method contained 80% to 85% pure CD14 ⁺ / CD33 ⁺ monocytes.

  A second monocyte population isolation method that differentiates cells based on density is due to differences in mobility during centrifugation. This method has certain advantages in clinical applications because it can use a disposable tubing set and can guarantee sterility and elimination of sample cross-contamination. Specifically, human blood samples were first processed to debulk red blood cells (RBCs) by centrifugal sedimentation using HESPan. The appropriate amount of 6% HESPan was then added to the anticoagulant treated blood until the final concentration reached 1.5%. The bag was mixed gently, incubated upright, and left at room temperature for 45 minutes to precipitate RBCs. The nucleated cell fraction (NCF) was then expressed using a manual plasma expresser and collected in another sterilized 600 mL blood bag. The resulting cell product was used as starting material for further separation based on density gradient centrifugation.

  Next, an Elutra® device (Gambro BCT Inc., Lakewood, Colorado) designed for enrichment of monocyte populations was utilized to process the starting cell product. The disposable tubing set was connected to Elutra®. Next, the first and second media bags containing the starting cell product, HBSS and 0.5% HSA were connected to the appropriate connecting part of the tubing set. The tubing set was primed using a second bag. Program number 1 (see Table 1 below) was used to process the starting cell product. The program automatically loaded the starting cell product into the chamber and processed using the first media bag. The cells were then continuously centrifuged and separated and collected in multiple fractions at various flow rates. The program was designed to collect 5 fractions each enriched with a unique cell population as follows. Platelet was collected in fraction 1, RBC in fraction 2, lymphocytes in fraction 3, monocytes in fraction 4 and granulocytes in fraction 5. Samples were taken from each fraction, analyzed for cell number and viability by nucleated cell count, and analyzed for purity by flow cytometry. Table 1 shows the flow rate and collected amount of each fraction. Based on purity and cell count, an appropriate amount containing monocytes was collected and then centrifuged at 300 g.

実施例２ 単離された単球集団による眼球血管傷害の治療 As shown in FIG. 2, monocyte preparations isolated by density centrifugation were found to be essentially similar to those isolated by FACS-based methods.

Example 2 Treatment of Ocular Vascular Injury with Isolated Monocyte Population

  An oxygen-induced retinopathy mouse model was used to test the therapeutic effects of monocyte populations isolated by the methods described herein. Mice with oxygen-induced retinopathy have been described by Ritter et al. , J. et al. Clin. Invet. 116: 3266-76. Specifically, oxygen-induced retinopathy is described in Smith et al. , Invest. Ophthalmol. Vis. Sci. 35: 101-111, 1994 and induced in C57BL / 6J mice. For comparison, BALB / cByJ mice were also exposed to the same conditions. Briefly, 7-day-old pups and their mother mice were transferred from room air to an environment of 75% oxygen for 5 days and then returned to room air. A high oxygen environment was created and maintained using a BioSpherix chamber. Under these conditions, in C57BL / 6J mice, a large oligovascular region was formed in the center of the retina during hyperoxia, and after returning to normoxia, abnormal pre-retinal neovascularization was caused, almost after birth. It peaked at 17 days and finally recovered.

The isolated cells were then injected intraocularly into mice. This is followed by immunohistochemical and visual analysis of the vasculature in the eyeballs of treated and control mice. These studies are described in Ritter et al. , J. et al. Clin. Invest. 116: 3266-76, 2006. The results of these studies are shown in FIG. 1B. As shown in the figure, the substantially pure population isolated by the present invention was able to promote vascular repair in mice suffering from oxygen-induced retinopathy.
Example 3 Other properties and activities of isolated monocyte populations

To demonstrate that the isolated cells are different in nature from other cell populations known in clinical use or clinical development, we have demonstrated that CD34 ⁺ cells, CD133 when expressed at high levels (ALDH ^br ). Peripheral blood samples were labeled for expression of aldehyde dehydrogenase identifying ⁺ cells, kit ⁺ cells, lineage antigen negative (Lin ⁻ ) cells. As a result, there is virtually no labeling in the peripheral blood sample (FIG. 3), which is consistent with the hypothesis that there will be very few stem cells in non-mobilized peripheral blood.

CD34 is a marker for hematopoietic stem cells and has been used to select cells for various clinical applications. The inventors have found that such cells may constitute the outcome of the therapeutic application described herein or may adversely affect it. Specifically, in order to identify the behavior of undifferentiated hematopoietic cells after intraocular injection, the inventors put mouse embryo cells and human mesenchymal stem cells (unlike CD34 ⁺ stem cells) into the vitreous body. Injected. These cells were injected into either normal eyeballs or oxygen-induced retinopathy (OIR) model eyeballs. In addition, to assess the effects of cell types not related to the eye, we injected normal human dermal fibroblasts into the vitreous of the OIR mouse model. In all the above cases, the inventors have found inflammatory and tumorigenic activity in the retina. These findings suggest that intraocular injection of undifferentiated stem cells and / or proliferating cells will lead to significant adverse events in normal or ischemic eyes. These studies show that, in contrast to undifferentiated stem cells, the myeloid progenitor cell population described in the present invention promotes controlled repair of the retinal vasculature without causing adverse events such as inflammation or tumorigenesis It also emphasizes the discovery of doing.

As shown in FIG. 4, a population prepared using the method of the present invention may contain a small number of CD34 ⁺ cells (FIG. 4). However, these cells are not necessary for the function of the model of the invention. Furthermore, the inventors selectively depleted CD34-expressing cells from the monocyte preparation of the present invention, but the effect was not changed.
Example 4 In Vitro Purity and Functional Assay of Isolated Monocytes

  In order to assess the purity and activity of monocytes isolated as described above, the inventors have developed several in vitro assays that independently evaluate the properties of various monocytes. The first assay was to measure the purity of the monocyte population. An antibody against the monocyte marker CD14 and flow cytometry were used (FIG. 5). As shown in FIG. 5, this assay allowed the inventors to quantify the number of non-monocytic cells present in the isolated cell population and to verify the efficiency of the isolation method of the present invention. . The second assay was an activity measurement of isolated monocytes. Cell chemotaxis to monocyte chemotaxis protein 1 (MCP-1) gradient was quantified. These tests were performed using a Boyden chamber with a pore size of 3 μm or 5 μm, where the cells were moved at 37 ° C. for 2 hours. The isolated monocyte preparation was found to migrate efficiently under the influence of MCP-1, but lymphocytes did not migrate (FIG. 6). Thus, this assay provided read-out information regarding the relative purity of the preparation and an indication of the viability and function of the isolated cells.

The third assay was based on the differential adhesion method to the cell culture base (cell separation method based on the difference in adhesion to the culture dish). It was established that monocytes can adhere to cell culture plastic while lymphocytes do not adhere. As evidenced in FIG. 7, the assay results demonstrated that the cells produced by the isolation methods described herein were primarily monocytes, as evidenced by their ability to adhere under these conditions.
Example 5 Systemic Administration of Treatment Cell Population

This example describes the intracardiac administration of CD44 ^hi bone marrow cells for therapeutic application in a retinopathy mouse model. This systemic route delivery is different from the typical topical route of administration (intraocular injection) used in the above examples. GFP-expressing CD44hi bone marrow cells are described in Ritter et al. , J. et al. Clin. Invest. 116: 3266-76, 2006. Intracardiac injections into C57BL / 6J mice (typically 7 day old mice) suffering from oxygen-induced retinopathy were performed using standard procedures. Vascular targeting activity of the cells was demonstrated by analyzing GS lectin stained retinas of mice several days after injection (eg, 7 or 10 days). Images of retinal vasculature were acquired using a Radiance 2100 MP confocal laser microscope (Bio-Rad; Zeiss). The procedure for retinal staining and analysis of confocal microscope images are described in Ritter et al. , J. et al. Clin. Invest. 116: 3266-76, 2006.

The results obtained from the study demonstrated that the fraction of treated cells targets the retina after hyperoxia injury (FIG. 8). These findings also allow for the systemic administration (eg, intracardiac injection) of the monocyte population described herein to obtain a therapeutic effect, such as restoring damage or delivering therapeutic substances to the eye. It is suggested that.
Example 6 Enhancing activity of an in vitro activated monocyte population

  This example describes the activation of a monocyte population ex vivo and its enhanced activity compared to non-activated cells.

  After isolating monocytic cells using the method described above, the isolated monocytic cells (fraction 5 (F5) cells) were treated with lipopolysaccharide (LPS) at a concentration of 25 ng / ml for 4 hours. Activity was measured by a flow cytometry-based assay, modified from the BD Intracellular Cytokine Stainin assay that measures intracellular levels of cytokines. This assay detects increased accumulation of IL-6, IL-8 and TNF proteins in monocytes (F5) cells treated with LPS compared to untreated cells and lymphocyte rich fraction (F3). It was done. Specifically, the data show that the lymphocyte-rich population (F3) is not substantially activated after LPS treatment, whereas the monocyte-rich population (F5) is clearly activated by LPS. Revealed. In addition, it was revealed that cells from diabetic donors were activated normally as measured by intracellular cytokine staining. Furthermore, flow cytometry analysis found that LPS treatment had little effect on F5 cell morphology when measured by forward scatter versus side scatter.

  The inventors independently verified these findings by using cytometric bead arrays to quantitatively measure cytokine levels secreted from LPS activated cells compared to untreated cells. The inventors have detected a significant increase in the secretion of IL-6, IL-8 and TNF proteins. This assay also confirmed that IL-1β was markedly up-regulated after LPS stimulation in F5 cells (FIG. 9), but IL-10 and IL-12p70 secretion were not substantially altered.

  In addition to activating isolated monocyte cells with LPS, the inventors also tested other activating compounds with more desirable safety properties. Specifically, the inventors initially focused their efforts on alternative ligands for the LPS receptor TLR4. One of these alternative TLR4 ligands, monophosphoryl lipid A (MPLA), was used in the cytometric bead array described above. The results, as found for LPS, MPLA activates F5 cells with increased IL-8, IL-6, and TNF (FIG. 10). An increase in IL-1 secreted β was also found after MPLA treatment, but the level was almost half that obtained by LPS stimulation.

  The inventors also tested the activation ability of mouse and human monocyte chemotactic protein 1 (MCP-1) on F5 monocyte cells. Both mouse and human MCP-1 stimulated increases in IL-8 and IL-6. However, the level was lower than that obtained with LPS or MPLA (FIGS. 10 and 11).

  In addition to measuring cytokine secretion in an ex vivo activated monocyte population, the inventors tested the therapeutic effects of cells in animal studies. In these studies, parallel groups of LPS-treated cells or control cells were administered to mice by intravitreal injection (250,000 cells in 0.5 μl). These animals were then exposed to hyperoxia and oxygen-induced retinopathy. The retinal analysis of these animals revealed two main parameters that were treated with LPS activated F5 monocytic cells: the area of vascular occlusion and the area of neovascularization (tufted branch). It was clarified to decrease. The decrease in these parameters was greater with LPS-treated F5 than with untreated F5 cells, treated F3 cells, vehicle or LPS alone.

  By using a value of 10,000 square microns below which the retina is considered substantially free of vascular occlusion (herein referred to as “healing”), the inventors After treatment with LPS-treated F5 cells compared to F5 or other LPS-treated fractions (F3), it was possible to prove that substantially more retinas had occluded regions below this cutoff (FIG. 12). ). This indicates that the activated cell population enriched in monocytes has the ability to promote vascular repair in a model of venous congestive retinopathy. As is apparent from FIG. 12, the F3 fraction shows a certain level of effect in the OIR model, suggesting that active cells are still present in this fraction. Thus, this population, or a combination of F5 and F3 cells, may also be therapeutically useful.

  With regard to the possibility of using autologous methods in the treatment of diabetic retinopathy, it is critical to prove that cells from diabetic donors are active. Using the OIR model, the inventors have shown that this is indeed the case. The monocyte-rich fraction from diabetic donors (F5) shows an activation pattern indistinguishable from normal donors (FIG. 9), these activated cells being non-activated F5 cells or other It was also revealed that vascular repair was promoted much more in the OIR model than the LTP treated fraction (F3) (FIG. 12). In addition, some activity was found in the F3 fraction rich in lymphocytes.

  The foregoing invention has been described in detail through explanations and examples for the sake of clarity, but certain changes and modifications will occur to those skilled in the art in light of the teachings of the invention. It will be apparent that the invention can be made without departing from the spirit and scope of the appended claims.

  All publications, databases, GenBank sequences, patents, and patent applications cited herein are hereby incorporated by reference herein as if each were specifically and individually indicated to be incorporated. Incorporated as part.

Claims (34)

  An isolated cell population comprising substantially pure monocytes that express the CD33 and CD14 antigens.   2. The isolated cell population of claim 1 isolated from a mammalian peripheral blood sample, umbilical cord blood sample or bone marrow sample.   2. The isolated cell population according to claim 1, wherein the cells in the isolated cell population are human cells or mouse cells.   2. The isolated cell population of claim 1, wherein at least 70%, 80% or 90% of the cells in the isolated cell population express the surface markers CD14 and CD33. CD34 ^- and it is claim 1 isolated cell population according. 2. The isolated cell population of claim 1, wherein the cell population is substantially free of ALDH ^br cells.   2. The isolated cell population of claim 1 further activated in vitro.   8. The method of claim 7, wherein the isolated cell population is activated by LPS, MPLA, or MCP-1.   Administering the isolated monocyte population to a patient suffering from an ocular vascular disorder, wherein the cell population administered here is in an amount sufficient to treat or ameliorate the ocular vasculopathy. A method of treating or ameliorating ocular vascular disorders.   10. The method of claim 9, wherein the monocyte population is isolated from a patient-derived blood sample or bone marrow sample.   The method of claim 9, wherein the patient is a human. 10. The method of claim 9, wherein the monocyte population comprises substantially pure CD14 ⁺ / CD33 ⁺ cells. 10. The method of claim 9, wherein at least 80% of the isolated monocyte population is CD14 ⁺ / CD33 ⁺ .   Claims wherein the ocular vascular disorder is selected from the group consisting of venous congestive retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlusion, retinal edema, macular degeneration and retinitis pigmentosa Item 10. The method according to Item 9.   10. The method of claim 9, wherein the monocyte population is administered to the patient via intravitreal injection.   10. The method of claim 9, wherein the monocyte population is activated in vitro or ex vivo before being administered to the patient.   17. The method of claim 16, wherein the monocyte population is activated by LPS, MPLA or MCP-1.   10. The method of claim 9, wherein the monocyte population is co-administered to the patient with a monocyte activating compound.   19. The method of claim 18, wherein the monocyte activating compound is LPS, MPLA, or MCP-1.   A method of treating or ameliorating a patient's ocular disease comprising: (i) isolating a substantially pure monocyte population from a blood sample or bone marrow sample of a patient suffering from ocular vascular disease; and (ii) Administering to the patient a monocyte population isolated in an amount sufficient to treat or ameliorate ocular vascular disease, thereby treating or ameliorating the symptoms of ocular vascular disease in the patient Method. 21. The method of claim 20, wherein the isolated monocyte population comprises substantially pure CD14 ⁺ / CD33 ⁺ cells.   21. The method of claim 20, wherein at least 80% of the isolated monocyte population expresses the surface markers CD33 and CD14.   21. The method of claim 20, wherein (i) debulking red blood cells from the sample; and (ii) residual red blood cells and other cell types in the sample based on their size, particle size or density The method wherein the monocyte population is isolated by separating from.   24. The method of claim 23, wherein residual red blood cells and other cell types are separated from monocytes by density centrifugation or fluorescence excited cell sorting (FACS).   21. The ocular vascular disorder is selected from the group consisting of venous congestive retinopathy, diabetic retinopathy, retinopathy of prematurity, neovascular glaucoma, central retinal vein occlusion, macular degeneration, and retinitis pigmentosa. The method described.   21. The method of claim 20, wherein the isolated monocyte population is activated ex vivo before being administered to the patient.   27. The method of claim 26, wherein the monocyte population is activated by LPS, MPLA or MCP-1.   A method of isolating a substantially pure monocyte population comprising: (i) providing a blood sample or bone marrow sample from a patient; (ii) debulking red blood cells from the sample; and (iii) in the sample Separating the remaining red blood cells and other cell types from the monocytes, thereby isolating a cell population comprising substantially pure monocytes.   29. The method of claim 28, wherein residual red blood cells and other cell types are separated from monocytes based on their size, particle size or density.   29. The method of claim 28, wherein residual red blood cells and other cell types are separated from monocytes by density centrifugation or fluorescence-excited cell sorting (FACS).   29. The method of claim 28, wherein the other cell types are platelets, granulocytes and granulocytes.   29. The method of claim 28, wherein the red blood cells are debulked by Hespan differential centrifugation or Ficoll density gradient centrifugation.   30. The method of claim 28, further comprising analyzing the isolated cell population for expression of the surface markers CD14 and CD33.   29. A substantially pure monocyte cell population isolated by the method of claim 28.

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