Therapeutic apoptotic cells for the treatment of osteoarthritis

Abstract

A method of use for treating osteoarthritis or vanishing bone disease in a subject in need thereof, the method comprising administering a composition of early apoptotic cells or apoptotic supernatant directly into or near the affected joint or bone tissue. The method of use can reduce pain, swelling, inflammation, bone loss and / or cartilage degeneration, and can increase the freedom of movement at the affected joint.

Description

[0001] Areas of public content

[0002] Disclosed herein are compositions and methods for treating osteoarthritis or vanishing bone disease in a subject. The methods disclosed herein include administering an early apoptotic cell composition directly into or near an affected joint, or near or adjacent to an affected bone tissue. Treatment methods include reducing pain, swelling, cartilage degeneration, and bone erosion.

[0003] background

[0004] The in vitro and in vivo properties of apoptotic cells indicate their potential use in a wide range of inflammatory and autoimmune conditions as well as conditions associated with cytokine storms. Apoptotic cells utilize a variety of mechanisms to generate an immune homeostatic anti-inflammatory state in macrophages and dendritic cells (DCs). These mechanisms include direct binding to phosphatidylserine (PtdSer) and indirect binding to Tyro3, ​​Axl, and Mer (TAM) receptors, as well as signal transduction through opsonins / bridging molecules, which use additional integrins and scavenger receptors (ScRs) to inhibit Toll-like receptors (TLRs) and NF-κb, STAT1, and IFN signal transduction, and activate liver X receptors (LXRs), cytokine signaling inhibitory proteins (Suppression of Cytokine Signaling, SOCS 1 / 3), peroxisome proliferator-activated receptors (PPAR)-δ, and hepatic growth factor (HGF). The sum of these events leads to downregulation, repair, and peripheral tolerance of inflammatory features of macrophages and DCs.

[0005] In fact, autoimmune and autoinflammatory conditions, including type 1 diabetes in non-obese diabetic mice, experimental autoimmune encephalomyelitis, arthritis, colitis, pulmonary fibrosis, fulminant hepatitis, contact hypersensitivity, acute and chronic graft rejection, hematopoietic cell transplantation, acute graft-versus-host disease (GvHD), and reduction of infarct size after acute myocardial infarction, have all been treated quite successfully by apoptotic cell infusion (Saas et al., (2016) "Concise Review: Apoptotic Cell-Based Therapies-Rationale, Preclinical Results and FutureClinical Developments." Stem Cells Volume 34(6):1464-1473). In most of the studies reviewed, apoptotic cells were administered systemically, with administration by intravenous or intraperitoneal injection (iv or ip, respectively) in studies specifically examining arthritis treatment.

[0006] Among the autoimmune and autoinflammatory conditions, the rheumatoid arthritis (RA) model is included in the review by Saas et al. (Saas et al., supra). In the collagen-induced arthritis (CIA) model, Gray et al. (M. Gray, K. Miles, D. Salter, D. Gray, and J. Savill. (2007) "Apoptotic cells protect mice from autoimmune inflammation by the induction of regulatory B cells." Proc Natl Acad Sci US A. Aug 28; 104 (35): 14080–14085.) identified the mechanism of action of RA. The data provided in Grey et al. show that in two different model systems and three different mouse strains, the administration of apoptotic cells can affect spleen B cells, causing them to secrete IL-10 and enhance the secretion of the immunosuppressive cytokine IL-10 from antigen-specific effector CD4+ T cells. In addition, apoptotic cells inhibited the severity of inflammatory arthritis and the production of pathogenic autoantibodies, despite immunization in the presence of the powerful adjuvant CFA. Therefore, the administration of apoptotic cells or passive transfer of B cells from AC-treated mice protected mice from CIA. Apoptotic cells themselves do not secrete immunosuppressive cytokines, such as IL-10 or TGF-β, and are not able to affect the progression of passively induced arthritis. This suggests that apoptotic cells can directly affect B cell function and induce cytokine production of regulatory B cells. In addition, in the CIA model, they found that injection of apoptotic cells by iv or ip route was protective because both delivered apoptotic cells to the spleen. The authors explicitly proposed that if the selected route of administration does not deliver apoptotic cells to the spleen so that apoptotic cells can interact with B cells, it is impossible to induce protection.

[0007] In another study using experimental streptococcal cell wall (SCW)-induced arthritis, Perruche et al. (2009) (Sylvain Perruche, Philippe Saas, and Wanjun Chen. "Apoptotic cell-mediated suppression of streptococcal cell wall-induced arthritis is associated with alteration of macrophage function and local regulatory T-cell increase: a potential cell-based therapy?" Arthritis Res Ther. 11(4): R104.) again demonstrated that the effects of apoptotic cells in the RA model were achieved through lymphoid organs and by the ip (equivalent to IV) administration route. They showed that only apoptotic cells administered by ip injection significantly suppressed the joint swelling and destruction commonly observed in the acute and chronic stages of SCW-induced arthritis. This is believed to be a result of higher Foxp3+Tregs in lymphoid organs, especially in draining lymph nodes.

[0008] Last but not least, Notley et al. (Notley CA1, Brown MA, Wright GP, Ehrenstein MR. (2011) "Natural IgM is required for suppression of inflammatory arthritis by apoptotic cells." J Immunol. April 15; 186 (8): 4967-72. doi: 10.4049 / jimmunol. 1003021.) used an Ag-induced inflammatory arthritis model to show that the enhanced production of IL-10 from T cells of draining lymph nodes and splenic marginal zone B cells driven by systemic infusion of apoptotic cells was abolished in the absence of natural IgM. Apoptotic cells were administered by iv injection and appeared in the splenic marginal zone shortly after administration.

[0009] The removal of dead cells is essential for rebuilding tolerance during inflammation and has an effective immunomodulatory effect. Since natural IgM plays a key role in removing apoptotic cells, Notely et al. (supra) studied whether the immunomodulatory properties of apoptotic cells depend on the presence of IgM. They used an inflammatory arthritis model that was unrelated to Ab and induced by Ag to test whether natural IgM was essential for the arthritis inhibitory properties of apoptotic cells. Although the administration of apoptotic cells reduced joint inflammation and damage in normal mice, accompanied by the inhibition of Th17 responses, it did not provide protection in secretory IgM-deficient (Sμ(-)) mice. The enhanced production of IL-10 from T cells and spleen marginal zone B cells in the draining lymph nodes driven by the infusion of apoptotic cells was eliminated in the absence of natural IgM. Apoptotic cells appeared in the spleen marginal zone shortly after administration, and it was inferred that natural IgM was a key factor in a series of events that promoted spleen IL-10 secretory B cells and T cells and inhibited the development of inflammation triggered by the administration of apoptotic cells.

[0010] Taken together, these results suggest that apoptotic cells improve RA and thereby autoimmunity through their effects on B and T cells in the spleen. No direct intra-articular effects are expected in non-autoimmune conditions.

[0011] Osteoarthritis is the most common musculoskeletal disorder, and there is currently no disease-modifying therapy available for osteoarthritis. Because knowledge about the development and maintenance of affected articular cartilage during osteoarthritis is still lacking, the current understanding of the disease mechanism of osteoarthritis is limited. During osteoarthritis, articular cartilage expresses markers of transient chondrogenic differentiation. In addition, blocking transient chondrogenic differentiation is sufficient to prevent the progression of experimental osteoarthritis. A developmental biology-inspired approach that combines the restoration of the tissue microenvironment, supplementation with engineered cartilage, and prevention of the intrinsic mechanism of transient chondrogenic differentiation may be a way to develop disease-modifying therapies for osteoarthritis. Nonsteroidal anti-inflammatory drugs that may relieve pain do not prevent the progressive degeneration of articular cartilage. Therefore, nonsteroidal anti-inflammatory drugs or corticosteroids are not considered to have therapeutic benefits for osteoarthritis, and in this regard, apoptotic cells are not really expected to improve osteoarthritis as immunomodulators.

[0012] "Vanishing bone disease" is a clinical manifestation that primarily involves the hip and shoulder but also other joints and may develop primarily from rapidly destructive arthritis caused by erosive osteoarthritis (Mavrogenis AF, Flevas DA, Panagopoulos GN et al. (2015) "Rapid destructive arthritis of the hip revisited." Eur J Orthop Surg Traumatol. 25: 1115-20) or spontaneous osteolysis caused by lymphangiomatous tissue proliferation, the Gorham-stout variant (Dellinger MT, Garg N, Olsen BR. (2014) "Viewpoints on vessels and vanishing bones in Gorham-Stout disease." Bone 63: 47-52). No effective treatment has been identified for these conditions.

[0013] Thus, there remains an unmet need for compositions and methods for treating non-autoimmune conditions such as osteoarthritis and vanishing bone disease, including treatments for reducing pain, reducing inflammation, reducing swelling, inhibiting or slowing the progressive degeneration of articular cartilage, inhibiting or slowing the erosion of bone tissue, and treatments for increasing motion, including increasing range of motion. Infusing apoptotic cells directly into joints may provide a new and safe treatment for rebalancing immune responses in bones and joints.

[0014] Overview

[0015] In one aspect, the present application discloses a method for treating osteoarthritis or vanishing bone disease in a subject in need thereof, the method comprising the step of administering a composition comprising an early apoptotic cell population or an apoptotic supernatant directly into a joint of the subject or at the site of vanishing bone disease, wherein the administration treats the subject's osteoarthritis or vanishing bone disease or a combination thereof.

[0016] In related aspects, when treating osteoarthritis, the treatment includes reducing pain, reducing inflammation, reducing swelling, inhibiting progressive degeneration of articular cartilage, reducing progressive degeneration of articular cartilage, improving quality of life, or any combination thereof. In related aspects, when treating vanishing bone disease, the treatment includes reducing pain, reducing inflammation, reducing swelling, inhibiting erosion of bone tissue, slowing erosion of bone tissue, reducing fractures, reducing bone damage, inhibiting fractures, inhibiting bone damage, inhibiting bone mass loss, inhibiting bone density loss, reducing bone mass loss, reducing bone density loss, or improving quality of life, or any combination thereof.

[0017] In another related aspect, a method of treating osteoarthritis increases motion of the joint, wherein the increased motion comprises increased range of motion or increased motion is accompanied by decreased pain, or a combination thereof.

[0018] In a related aspect, the joint comprises a synovial joint. In another related aspect, the synovial joint comprises a knee joint, a hip joint, a shoulder joint, a joint between cervical vertebrae, an elbow joint, an ankle joint, a wrist joint, a finger joint, a toe joint, or a thumb joint, a hand joint, a foot joint, or a combination thereof.

[0019] In related aspects, the site of vanishing bone disease includes the shoulder, skull, pelvic girdle, jaw, one or more ribs, clavicle, or spine, or a combination thereof.

[0020] In related aspects of the treatment methods disclosed herein, direct administration into the joint comprises infusion or injection of the early apoptotic cell population or the apoptotic supernatant.

[0021] In a related aspect of the method of treatment disclosed herein, the early apoptotic cell population includes an autologous early apoptotic cell population that is stable for more than 24 hours; or an allogeneic early apoptotic cell population that is stable for more than 24 hours. In another related aspect, the apoptotic supernatant is collected from cultured apoptotic cells, cultured early apoptotic cells, or apoptotic cells cultured with leukocytes. In yet another related aspect, the supernatant can be pooled. In yet another related aspect, the early apoptotic cell population includes a pooled early apoptotic cell population. In another related aspect, the pooled early apoptotic cell population includes a irradiated, pooled early apoptotic cell population. In another related aspect, the pooled early apoptotic cell population includes apoptotic cells prepared from a single donor mononuclear cell or from multiple donor mononuclear cells. In another related aspect, the early apoptotic cell population includes a irradiated early apoptotic cell population.

[0022] In related aspects of the therapeutic methods disclosed herein, the subject is a human subject.

[0023] In a related aspect of the methods of treatment disclosed herein, administration comprises a single administration of the early apoptotic cell population or the apoptotic supernatant. In another related aspect, administration comprises more than one administration of the apoptotic cell population or the apoptotic supernatant. In yet another related aspect, more than one administration comprises daily or weekly administration.

[0024] In related aspects of the methods of treatment disclosed herein, each administered dose comprises about 1×10 6 ±20% to 1×10 9± 20% of early apoptotic cells / kg of subject. In another related aspect, each dose administered comprises a dose of about 1×10 6 ±20% to 1×10 9 ±20% apoptotic cells / kg of subject were collected from the culture supernatants.

[0025] In related aspects of the treatment methods disclosed herein, administration reduces the concentration of at least one pro-inflammatory or anti-inflammatory cytokine or chemokine in synovial fluid present in a joint. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the concluding portions of the specification. However, the organization and method of operation of the invention, together with objects, features, and advantages thereof, may be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings, in which:

[0028] Figure 1 A flow chart of steps in some embodiments of a method for preparing a population of early apoptotic cells is presented, wherein an anticoagulant is included in the method (see Examples 1 and 2 for details of different embodiments). The collected mononuclear cells can be autologous or allogeneic, wherein in some embodiments mismatched mononuclear cells are used. If more than one cell source is used, additional steps include irradiating the cells and pooling the mismatched cells.

[0029] Figure 2 A series of bar graphs are presented showing cytokine / chemokine levels in the right shoulder joint before and after treatment with early apoptotic cells. Cytokine / chemokine measurements were performed by the Luminex MAGPIX system and analyzed using Milliplex software (see Example 4 below).

[0030] Detailed Description

[0031] In the following detailed description, many specific details are set forth to provide a thorough understanding of early apoptotic cell populations and their use for treating osteoarthritis. However, those skilled in the art will appreciate that the use of early apoptotic cell populations or compositions thereof can be practiced without these specific details. In other cases, well-known methods, procedures, and components are not described in detail to avoid obscuring the present disclosure.

[0032] In some embodiments, disclosed herein are methods of treating osteoarthritis or vanishing bone disease, or a combination thereof, in a subject in need thereof, the method comprising the step of administering a composition comprising an early apoptotic cell population directly into a joint of the subject or at the site of vanishing bone disease, wherein the administration treats osteoarthritis or vanishing bone disease in the subject. In other embodiments, disclosed herein are methods of treating osteoarthritis or vanishing bone disease, or a combination thereof, in a subject in need thereof, the method comprising the step of administering a composition comprising apoptotic cell supernatant directly into a joint of the subject or at the site of vanishing bone disease, wherein the administration treats osteoarthritis or vanishing bone disease in the subject.

[0033] Unlike rheumatoid arthritis, osteoarthritis and vanishing bone disease are not considered inflammatory autoimmune diseases or disorders. The methods described herein relate to a variety of treatment methods and are not limited to treating autoimmunity. In some embodiments, the methods described herein bypass the necessity of the systemic immune system and any of its responses as an essential element for effective treatment. In some embodiments, treatment is performed by administering a population of early apoptotic cells or a composition thereof directly to the site of osteoarthritis or vanishing bone disease. In some embodiments, treatment is performed by administering apoptotic cell supernatants or a composition thereof directly to the site of osteoarthritis or vanishing bone disease.

[0034] Apoptotic cells

[0035] The generation of early apoptotic cells ("ApoCell") used in the compositions and methods as disclosed herein has been described in WO2014 / 087408 (which is incorporated herein by reference in its entirety) and is briefly described in Example 1 below. In another embodiment, the early apoptotic cells used in the compositions and methods disclosed herein are produced by any method known in the art. In another embodiment, the early apoptotic cells used in the compositions and methods disclosed herein are autologous to the subject being treated. In another embodiment, the early apoptotic cells used in the compositions and methods disclosed herein are allogeneic to the subject being treated. In another embodiment, the early apoptotic cells used in the compositions and methods disclosed herein are not matched to the subject being treated, in other words, they are "off the shelf" (OTS). In another embodiment, the composition comprising early apoptotic cells comprises early apoptotic cells disclosed herein or known in the art

[0036] The skilled artisan will understand that the term "autologous" may include tissues, cells, nucleic acid molecules or polypeptides in which the donor and recipient are the same individual.

[0037] The skilled artisan will understand that the term "allogeneic" can include tissues, cells, nucleic acid molecules or polypeptides derived from different individuals of the same species. In some embodiments, the allogeneic donor cells are genetically different from the recipient.

[0038] In some embodiments, the cell source of the early apoptotic population includes enriched mononuclear cells. In some embodiments, the cell composition (mononuclear-enriched cell composition) enriched in mononuclear cells is obtained according to the production method disclosed herein, which is achieved by leukapheresis. The technician will understand that the term "leukapheresis" can include a single collection procedure, in which leukocytes are separated from the blood of the donor. In some embodiments, the blood of the donor undergoes leukapheresis, and therefore the cell composition enriched in mononuclear cells is obtained according to the production method disclosed herein. It should be noted that, as known in the art, at least one anticoagulant needs to be used during leukapheresis to prevent the collected cells from condensing.

[0039] In some embodiments, according to the production method disclosed herein, the leukocyte apheresis procedure is configured to allow the collection of cell compositions enriched in mononuclear cells. In some embodiments, the cell collection obtained by leukocyte apheresis includes at least 65% mononuclear cells. In other embodiments, as disclosed herein, at least 70% or at least 80% mononuclear cells. In some embodiments, according to the production method disclosed herein, while obtaining a cell composition enriched in mononuclear cells, plasma from a cell donor is collected. In some embodiments, according to the production method disclosed herein, while obtaining a cell composition enriched in mononuclear cells, about 300-600ml plasma from a cell donor is collected. In some embodiments, according to the production method disclosed herein, plasma is collected while obtaining a cell composition enriched in mononuclear cells, and is used as a part of freezing and / or incubation medium. Other detailed methods for obtaining a mononuclear population of enriched apoptotic cells for the compositions and methods disclosed herein can be found in WO 2014 / 087408 (which is incorporated herein by reference in its entirety).

[0040] In some embodiments, the early apoptotic cells used in the methods disclosed herein comprise at least 85% mononuclear cells. In other embodiments, the early apoptotic cells used in the methods disclosed herein contain at least 85% mononuclear cells, 90% mononuclear cells, or alternatively more than 90% mononuclear cells. In some embodiments, the early apoptotic cells used in the methods disclosed herein comprise at least 90% mononuclear cells. In some embodiments, the early apoptotic cells used in the methods disclosed herein comprise at least 95% mononuclear cells.

[0041] It should be noted that in some embodiments, while the mononuclear-enriched cell preparation comprises at least 65%, preferably at least 70%, and most preferably at least 80% mononuclear cells at the time of cell collection, the final pharmaceutical population comprises at least 85%, preferably at least 90%, and most preferably at least 95% mononuclear cells following the method for generating early apoptotic cells used in the methods disclosed herein.

[0042] In certain embodiments, the cell preparation of enriched mononuclear cells for producing the early apoptotic cell composition for the method disclosed herein comprises at least 50% mononuclear cells at the time of cell collection. In certain embodiments, disclosed herein is a method for producing a pharmaceutical population, wherein the method comprises obtaining a cell preparation of enriched mononuclear cells from the peripheral blood of a donor, the cell preparation of enriched mononuclear cells comprising at least 50% mononuclear cells. In certain embodiments, disclosed herein is a method for producing a pharmaceutical population, wherein the method comprises freezing a cell preparation of enriched mononuclear cells comprising at least 50% mononuclear cells.

[0043] In some embodiments, the cell preparation comprises at least 85% mononuclear cells, wherein at least 40% of the cells in the preparation are in an early apoptotic state, and wherein at least 85% of the cells in the preparation are viable cells. In some embodiments, the apoptotic cell preparation comprises no more than 15% CD15 高 Expressing cells.

[0044] The skilled person will understand that the term "early apoptotic state" can include cells that show early signs of apoptosis but not late signs of apoptosis. Examples of early signs of apoptosis in cells include exposure of phosphatidylserine (PS) and loss of mitochondrial membrane potential. Examples of late events include entry of propidium iodide (PI) into cells and final DNA cleavage. In order to record that cells are in an "early apoptotic" state, in some embodiments, PS exposure and PI staining by Annexin-V are used, and cells stained by Annexin-V but not stained by PI or only stained with very low PI are considered "early apoptotic cells" (An + PI -). In some embodiments, very low IP staining includes less than or equal to (≤) 15% PI+ cells in a cell population. In some embodiments, very low IP staining includes ≤10% PI+ cells in a cell population. In some embodiments, very low IP staining includes ≤5% PI+ cells in a cell population. In another embodiment, cells stained with Annexin-V FITC and high PI are considered "late apoptotic cells". In some embodiments, high IP staining includes greater than (>) 15% PI+ cells in a cell population. In some embodiments, high IP staining includes greater than or equal to (≥) 16% PI+ cells in a cell population. In another embodiment, cells not stained with Annexin-V or PI are considered non-apoptotic viable cells.

[0045] In some embodiments, at least 40% of the cells in the product are in an early apoptotic state. In some embodiments, at least 45% of the cells in the product are in an early apoptotic state. In some embodiments, at least 50% of the cells in the product are in an early apoptotic state. In some embodiments, at least 55% of the cells in the product are in an early apoptotic state. In some embodiments, at least 60% of the cells in the product are in an early apoptotic state. In some embodiments, at least 65% of the cells in the product are in an early apoptotic state. In some embodiments, at least 70% of the cells in the product are in an early apoptotic state. In some embodiments, at least 75% of the cells in the product are in an early apoptotic state. In some embodiments, at least 80% of the cells in the product are in an early apoptotic state. In some embodiments, at least 85% of the cells in the product are in an early apoptotic state. In some embodiments, at least 90% of the cells in the product are in an early apoptotic state. In some embodiments, at least 95% of the cells in the product are in an early apoptotic state.

[0046] In some embodiments, the early apoptotic cell preparation comprises less than or equal to (≤) 15% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 10% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 9% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 8% PI + In some embodiments, the early apoptotic cell preparation comprises ≤7% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 6% PI + In some embodiments, the preparation of early apoptotic cells comprises ≤5% PI + In some embodiments, the early apoptotic cell preparation comprises ≤4% PI +In some embodiments, the early apoptotic cell preparation comprises ≤ 3% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 2% PI + In some embodiments, the early apoptotic cell preparation comprises ≤ 1% PI + cell.

[0047] In some embodiments, at least 40% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 45% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 50% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 55% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 60% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 65% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 70% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 75% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI +In some embodiments, at least 80% of the cells in the preparation are in an early apoptotic state (An + ), of which ≤15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 85% of the cells in the preparation are in an early apoptotic state (An + ), of which <15% or ≤14%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of the cells were PI + In some embodiments, at least 90% of the cells in the preparation are in an early apoptotic state (An + ), of which <10% or ≤9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0% of cells were PI + In some embodiments, at least 95% of the cells in the preparation are in an early apoptotic state (An + ), of which <5%, ≤4%, 3%, 2%, 1% or 0% of cells were PI + .

[0048] The skilled artisan will understand that in some embodiments, the terms "apoptotic cells", "early apoptotic cells", "Allocetra", "Autocetra", "ALC" and "ApoCell" and grammatical variations thereof can be used interchangeably to refer to a population of "early apoptotic cells", wherein the cell population is enriched for mononuclear cells and has unique characteristics (see, e.g., Example 1).

[0049] In some embodiments, Allocetra comprises an early apoptotic cell population obtained from a single allogeneic donor. In some embodiments, Allocetra comprises an early apoptotic cell population obtained from more than one allogeneic donor. In some embodiments, Allocetra includes a pooled early apoptotic cell population obtained from more than one allogeneic donor or from cells from a blood bank. In some embodiments, Allocetra includes a pooled early apoptotic cell population obtained from the same allogeneic donor. In some embodiments, Allocetra comprises an irradiated early apoptotic cell population. In some embodiments, the term "Allocetra" can be used interchangeably with the term "Allocetra-OTS". In some embodiments, the terms "Allocetra" and "Allocetra-OTS" include mononuclear early apoptotic cells, prepared as described in Example 1, regardless of the source of the cells.

[0050] In some embodiments, Autocetra includes a population of early apoptotic cells obtained from a single donation from an autologous donor. In some embodiments, Autocetra includes a population of early apoptotic cells obtained from more than one donation from an autologous donor. In some embodiments, the early apoptotic cells from an autologous donor used in a method for treating osteoarthritis or vanishing bone disease are irradiated. In some embodiments, Autocetra includes a population of irradiated early apoptotic cells. In some embodiments, the term "Autocetra" can be used interchangeably with the term "Autocetra-OTS".

[0051] It will be appreciated by the skilled artisan that in some embodiments, the compositions and methods described herein comprise early apoptotic cells. In some embodiments, as described herein, early apoptotic cells are HLA matched with the recipient (subject in need) of the composition comprising early apoptotic cells. In some embodiments, as described herein, early apoptotic cells are mismatched with the recipient (subject in need) of the composition comprising early apoptotic cells. In some embodiments, early apoptotic cells that are mismatched with the recipient (subject in need) of the composition comprising early apoptotic cells are irradiated as described in detail herein. In some embodiments, irradiated mismatched cells are referred to as "Allocetra-OTS" or "ALC-OTS".

[0052] In some embodiments, apoptotic cells include cells in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 90% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 80% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 70% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 60% of the cells are in an early apoptotic state. In another embodiment, apoptotic cells include cells in which at least 50% of the cells are in an early apoptotic state.

[0053] In some embodiments, the composition comprising apoptotic cells further comprises an anticoagulant.

[0054] In some embodiments, the early apoptotic cells are stable. The skilled artisan will appreciate that in some embodiments, stability includes maintaining early apoptotic cell characteristics over time, for example, maintaining early apoptotic cell characteristics when stored at about 2-8° C. In some embodiments, stability includes maintaining early apoptotic cell characteristics when stored at a frozen temperature (e.g., at a temperature at or below 0° C.).

[0055] In some embodiments, the cell population (mononuclear-enriched cell population) of enriched mononuclear cells obtained according to the method for producing early apoptotic cells of the methods disclosed herein is subjected to freezing in a freezing medium. In some embodiments, freezing is gradual. In some embodiments, after collecting the cells, they are kept at room temperature until freezing. In some embodiments, the cell product undergoes at least one washing step in a washing medium after the cells are collected and before freezing.

[0056] As used herein, the terms "obtaining cells" and "cell collection" can be used interchangeably. In some embodiments, the cells of the cell product are frozen within 3-6 hours after collection. In some embodiments, the cell product is frozen within a maximum of 6 hours after the cell is collected. In some embodiments, the cells of the cell product are frozen within 1, 2, 3, 4, 5, 6, 7, 8 hours after collection. In other embodiments, the cells of the cell product are frozen at most 8, 12, 24, 48, 72 hours after collection. In other embodiments, after the cells are collected, they are kept at 2-8°C until frozen.

[0057] In some embodiments, freezing according to the generation of an early apoptotic cell population includes freezing the cell preparation at about -18°C to -25°C, then freezing the cell preparation at about -80°C, and finally freezing the cell preparation in liquid nitrogen until thawing. In some embodiments, freezing according to the generation of an early apoptotic cell population includes freezing the cell preparation at about -18°C to -25°C for at least 2 hours, freezing the cell preparation at about -80°C for at least 2 hours, and finally freezing the cell preparation in liquid nitrogen until thawing. In some embodiments, the cells are stored in liquid nitrogen for at least 8, 10 or 12 hours before thawing. In some embodiments, the cells of the cell preparation are stored in liquid nitrogen until thawing and incubated with an apoptosis-inducing incubation medium. In some embodiments, the cells of the cell preparation are stored in liquid nitrogen until the day of hematopoietic stem cell transplantation. In a non-limiting example, the time from cell collection and freezing to the preparation of the final population can be between 1-50 days, optionally between 6-30 days. In an optional embodiment, the cell preparation can be stored in liquid nitrogen for a longer time, such as at least several months.

[0058] In some embodiments, freezing in accordance with the generation of an early apoptotic cell population comprises freezing the cell preparation at about -18°C to -25°C for at least 0.5, 1, 2, 4 hours. In some embodiments, freezing in accordance with the generation of an early apoptotic cell population comprises freezing the cell preparation at about -18°C to -25°C for about 2 hours. In some embodiments, freezing in the generation of an early apoptotic cell population comprises freezing the cell preparation at about -80°C for at least 0.5, 1, 2, 4, 12 hours.

[0059] In some embodiments, the cell composition enriched for mononuclear cells can be kept frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 20 months. In some embodiments, the cell composition enriched for mononuclear cells can be kept frozen for at least 0.5, 1, 2, 3, 4, 5 years. In certain embodiments, the cell composition enriched for mononuclear cells can be kept frozen for at least 20 months.

[0060] In some embodiments, the cell composition enriched in mononuclear cells is frozen for at least 8, 10, 12, 18, 24 hours. In certain embodiments, the cell composition enriched in mononuclear cells is frozen for a period of at least 8 hours. In some embodiments, the cell composition enriched in mononuclear cells is frozen for at least about 10 hours. In some embodiments, the cell composition enriched in mononuclear cells is frozen for at least about 12 hours. In some embodiments, the cell composition enriched in mononuclear cells is frozen for about 12 hours. In some embodiments, the total freezing time of the cell composition enriched in mononuclear cells (at about -18 ° C to -25 ° C, at about -80 ° C and in liquid nitrogen) is at least 8, 10, 12, 18, 24 hours.

[0061] In some embodiments, in the cells of the cell composition enriched with mononuclear cells, freezing at least partially induces an early apoptotic state. In some embodiments, the freezing medium comprises RPMI1640 culture medium, which comprises L-glutamine, Hepes, Hes, dimethyl sulfoxide (DMSO) and plasma. In some embodiments, the plasma in the freezing medium is the autologous plasma of the donor of the cells enriched with mononuclear cells in the donation population. In some embodiments, the freezing medium comprises RPMI1640 culture medium, which comprises 2mM L-glutamine, 10mM Hepes, 5% Hes, 10% dimethyl sulfoxide and 20% v / v plasma.

[0062] In some embodiments, the freezing medium comprises an anticoagulant. In certain embodiments, at least some of the media (including freezing media, incubation media, and wash media) used in the production process of the early apoptotic cell colony comprises an anticoagulant. In certain embodiments, all media containing anticoagulants used in the production process of the early apoptotic cell colony comprise the same concentration of anticoagulant. In some embodiments, no anticoagulant is added to the final suspension medium of the cell colony.

[0063] In some embodiments, at least adding an anticoagulant to the freezing medium increases the yield of the cell product. In other embodiments, adding an anticoagulant to the freezing medium increases the yield of the cell product in the presence of high triglyceride levels. As used herein, the increase in the yield of the cell product relates to an increase in at least one of the following: the percentage of surviving cells in the frozen cells, the percentage of early-stage apoptotic cells in the surviving cells, and combinations thereof.

[0064] In some embodiments, early apoptotic cells are stable for at least 24 hours. In another embodiment, early apoptotic cells are stable for 24 hours. In another embodiment, early apoptotic cells are stable for more than 24 hours. In another embodiment, early apoptotic cells are stable for at least 36 hours. In another embodiment, early apoptotic cells are stable for 48 hours. In another embodiment, early apoptotic cells are stable for at least 36 hours. In another embodiment, early apoptotic cells are stable for more than 36 hours. In another embodiment, early apoptotic cells are stable for at least 48 hours. In another embodiment, early apoptotic cells are stable for 48 hours. In another embodiment, early apoptotic cells are stable for at least 48 hours. In another embodiment, early apoptotic cells are stable for more than 48 hours. In another embodiment, early apoptotic cells are stable for at least 72 hours. In another embodiment, early apoptotic cells are stable for 72 hours. In another embodiment, early apoptotic cells are stable for more than 72 hours.

[0065] Technicians will understand that the term "stable" includes apoptotic cells that maintain PS positive (phosphatidylserine positive), only a very small percentage of PI positive (propidium iodide positive). PI positive cells provide an indication of membrane stability, wherein PI positive cells allow entry into cells indicating that the membrane is less stable. In some embodiments, stable early apoptotic cells remain in an early apoptotic state for at least 24 hours, at least 36 hours, at least 48 hours, or at least 72 hours. In another embodiment, stable early apoptotic cells remain in an early apoptotic state for 24 hours, 36 hours, 48 ​​hours, or 72 hours. In another embodiment, stable early apoptotic cells remain in an early apoptotic state for more than 24 hours, more than 36 hours, more than 48 hours, or more than 72 hours. In another embodiment, stable early apoptotic cells maintain their state for a period of time that continues to extend.

[0066] In some embodiments, the apoptotic cell population is free of cell aggregates. In some embodiments, the apoptotic cell population is free of large cell aggregates. In some embodiments, the apoptotic cell population has a reduced number of cell aggregates compared to an apoptotic cell population prepared without adding an anticoagulant in a step other than collecting cells from a donor (leukocytapheresis). In some embodiments, the apoptotic cell population or a composition thereof comprises an anticoagulant.

[0067] In some embodiments, the apoptotic cells are free of cell aggregates, wherein the apoptotic cells are obtained from a subject with high blood triglycerides. In some embodiments, the subject's blood triglyceride level is above 150 mg / dL. In some embodiments, the apoptotic cell population is free of cell aggregates, wherein the apoptotic cell population is prepared from cells obtained from a subject with normal blood triglycerides. In some embodiments, the subject's blood triglyceride level is equal to or less than 150 mg / dL. In some embodiments, during the apoptotic cell generation method, cell aggregates produce cell loss.

[0068] The skilled artisan will appreciate that the term "aggregate" or "cell aggregate" may include reversible coagulation of blood cells under low shear or static conditions. During the incubation step to produce apoptotic cells, cell aggregates may be visually observed. Cell aggregation may be measured by any method known in the art, such as by visually imaging the sample under an optical microscope or using flow cytometry.

[0069] In some embodiments, the anticoagulant is selected from the group consisting of heparin, acid citrate dextrose (ACD) Formula A, and combinations thereof. In some embodiments, the anticoagulant is selected from the group consisting of heparin, acid citrate dextrose (ACD) Formula A, and combinations thereof.

[0070] In some embodiments of the method for preparing an early apoptotic cell population and its composition, an anticoagulant is added to at least one medium used in the process of preparing the population. In some embodiments, at least one medium used in the process of preparing the population is selected from the group consisting of: freezing medium, washing medium, apoptosis induction incubation medium and any combination thereof.

[0071] In some embodiments, the anticoagulant is selected from the group consisting of: heparin, ACD formula A, and combinations thereof. It should be noted that other anticoagulants known in the art may be used, such as, but not limited to, Fondaparinaux, Bivalirudin, and Argatroban.

[0072] In some embodiments, at least one medium used in the preparation of the population contains 5% ACD Formula A solution containing 10 U / ml heparin. In some embodiments, no anticoagulant is added to the final suspension medium of the cell population. As used herein, the terms "final suspension medium" and "administration medium" are used interchangeably and have all the same properties and meanings.

[0073] In some embodiments, at least one medium used in the preparation of the population comprises heparin at a concentration between 0.1-2.5 U / ml. In some embodiments, at least one medium used during the preparation of the population comprises ACD formula A at a concentration between 1%-15% v / v. In some embodiments, the freezing medium comprises an anticoagulant. In some embodiments, the incubation medium comprises an anticoagulant. In some embodiments, both the freezing medium and the incubation medium comprise an anticoagulant. In some embodiments, the anticoagulant is selected from the group consisting of heparin, ACD formula A, and combinations thereof.

[0074] In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U / ml. In some embodiments, the concentration of ACD formula A in the freezing medium is between 1%-15% v / v. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U / ml. In some embodiments, the concentration of ACD formula A in the incubation medium is between 1%-15% v / v. In some embodiments, the anticoagulant is a citrate dextrose (ACD) formula A solution. In some embodiments, the anticoagulant added to at least one medium used in the preparation of the population is an ACD formula A containing heparin at a concentration of 10U / ml.

[0075] In some embodiments, the apoptosis-inducing incubation medium for producing an early apoptotic cell colony comprises an anticoagulant. In some embodiments, the freezing medium and the apoptosis-inducing incubation medium for producing an early apoptotic cell colony both comprise an anticoagulant. Without wishing to be bound by any theory or mechanism, in order to maintain a high and stable cell yield in different cell compositions, regardless of the cell collection scheme, in some embodiments, the addition of an anticoagulant includes adding an anticoagulant to both the freezing medium and the apoptosis-inducing incubation medium during the generation of the apoptotic cell colony. In some embodiments, the high and stable cell yield in the composition includes at least 30%, preferably at least 40%, and usually at least 50% of the cell yield in the initial cell colony for inducing apoptosis.

[0076] In some embodiments, both the freezing medium and the incubation medium include an anticoagulant. In some embodiments, regardless of the cell collection conditions, such as but not limited to the timing and / or type of the anticoagulant added during cell collection, adding an anticoagulant to both the incubation medium and the freezing medium results in a high and stable cell yield of different population products. In some embodiments, regardless of the timing and / or type of adding an anticoagulant during leukocyte apheresis, adding an anticoagulant to both the incubation medium and the freezing medium results in a high and stable yield of cell products. In some embodiments, producing cell products in the presence of high triglyceride levels results in low and / or unstable cell yields between different products. In some embodiments, producing cell products from donor blood with high triglyceride levels results in low and / or unstable cell yields of cell products. In some embodiments, the term "high triglyceride levels" refers to triglyceride levels higher than the normal levels of healthy subjects of the same sex and age. In some embodiments, the term "high triglyceride levels" refers to triglyceride levels higher than about 1.7mM / liters. As used herein, a high and stable yield refers to a cell yield in a population that is high enough to enable the preparation of a dose that will show therapeutic efficacy when administered to a subject. In some embodiments, therapeutic efficacy refers to the ability to treat, prevent, or improve an immune disease, autoimmune disease, or inflammatory disease in a subject. In some embodiments, a high and stable cell yield is a cell yield of at least 30%, possibly at least 40%, and usually at least 50% of the cells in a population of cells that were initially frozen.

[0077] In some embodiments, where the cell product is obtained from a donor with high triglyceride levels, the donor will take at least one measure selected from the group consisting of: taking a triglyceride-lowering medication such as, but not limited to, statins and / or bezafibrate prior to donation, fasting for a period of at least 8, 10, 12 hours prior to donation, eating an appropriate diet to lower blood triglyceride levels at least 24, 48, 72 hours prior to donation, and any combination thereof.

[0078] In some embodiments, the cell yield in a population refers to the number of cells of the composition / the initial number of cells that undergo apoptosis induction.As used herein, the terms "induce an early apoptotic state" and "induce apoptosis" may be used interchangeably.

[0079] In some embodiments, after freezing and thawing, the cell composition enriched for mononuclear cells is incubated in an incubation medium. In some embodiments, there is at least one washing step between thawing and incubation. As used herein, the terms "incubation medium" and "apoptosis-inducing incubation medium" are used interchangeably. In some embodiments, the incubation medium comprises RPMI 1640 culture medium supplemented with L-glutamine, Hepes, methylprednisolone and plasma. In some embodiments, the washing medium comprises 2mM L-glutamine, 10mM Hepes and 10% v / v plasma. In some embodiments, the plasma in the incubation medium is derived from the same donor from which the cells of the cell product are obtained. In some embodiments, plasma is added to the incubation medium on the day of incubation. In some embodiments, incubation is carried out at 37°C and 5% CO2.

[0080] In some embodiments, the incubation medium comprises methylprednisolone. In some embodiments, the methylprednisolone in the incubation medium further induces the cells in the cell composition enriched for mononuclear cells to enter an early apoptotic state. In some embodiments, the cells in the cell composition enriched for mononuclear cells are induced to enter an early apoptotic state by freezing and incubating both in the presence of methylprednisolone. In some embodiments, the generation of an early apoptotic cell population advantageously allows induction of a significant early apoptotic state without substantially inducing necrosis, wherein the prepared cells remain stable in the early apoptotic state for about 24 hours.

[0081] In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 10-100 μg / ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of about 40-60 μg / ml, optionally about 45-55 μg / ml. In some embodiments, the incubation medium comprises methylprednisolone at a concentration of 50 μg / ml.

[0082] In some embodiments, incubation lasts for about 2-12 hours, may last for 4-8 hours, and typically lasts for about 5-7 hours. In some embodiments, incubation lasts for about 6 hours. In some embodiments, incubation lasts for at least 6 hours. In preferred embodiments, incubation lasts for 6 hours.

[0083] In some embodiments, the incubation medium comprises an anticoagulant. In some embodiments, adding an anticoagulant to the incubation medium increases the yield of the cell product. In some embodiments, the concentration of the anticoagulant in the incubation medium is the same as the concentration in the freezing medium. In some embodiments, the incubation medium comprises an anticoagulant selected from the group consisting of: heparin, ACD formula A, and combinations thereof. In some embodiments, the anticoagulant used in the incubation medium is an ACD formula A containing heparin at a concentration of 10U / ml.

[0084] In some embodiments, the incubation medium comprises heparin. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U / ml. In some embodiments, the concentration of heparin in the incubation medium is between 0.1-2.5U / ml, possibly between 0.3-0.7U / ml, and generally about 0.5U / ml. In certain embodiments, the concentration of heparin in the incubation medium is about 0.5U / ml.

[0085] In some embodiments, the incubation medium comprises ACD Formulation A. In some embodiments, the concentration of ACD Formulation A in the medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formulation A in the incubation medium is between 1%-15% v / v, may be between 4%-7% v / v, and is typically about 5% v / v. In some embodiments, the concentration of ACD Formulation A in the incubation medium is about 5% v / v.

[0086] In some embodiments, the increase in the yield of a cell preparation comprises an increase in the number of early apoptotic viable cells in the preparation / the number of frozen cells from which the preparation is prepared.

[0087] In some embodiments, adding an anticoagulant to the freezing medium contributes to high and stable yields between different preparations of the drug population. In a preferred embodiment, regardless of the cell collection scheme used, adding an anticoagulant to at least the freezing medium and the incubation medium results in high and stable yields between different pharmaceutical composition preparations.

[0088] In some embodiments, the freezing medium comprises an anticoagulant selected from the group consisting of: heparin, ACD formula A, and combinations thereof. In some embodiments, the anticoagulant used in the freezing medium is ACD formula A containing heparin at a concentration of 10 U / ml. In some embodiments, the freezing medium comprises a 5% v / v ACD formula A solution containing heparin at a concentration of 10 U / ml.

[0089] In some embodiments, the freezing medium comprises heparin. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U / ml. In some embodiments, the concentration of heparin in the freezing medium is between 0.1-2.5U / ml, possibly between 0.3-0.7U / ml, and generally about 0.5U / ml. In certain embodiments, the concentration of heparin in the freezing medium is about 0.5U / ml.

[0090] In some embodiments, the cryoincubation medium comprises ACD Formula A. In some embodiments, the concentration of ACD Formula A in the freezing medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formula A in the freezing medium is between 1%-15% v / v, possibly between 4%-7% v / v, and typically about 5% v / v. In some embodiments, the concentration of ACD Formula A in the freezing medium is about 5% v / v.

[0091] In some embodiments, regardless of the triglyceride level in the donor blood, adding an anticoagulant to the incubation medium and / or the freezing medium results in a high and stable cell yield in the population. In some embodiments, adding an anticoagulant to the incubation medium and / or the freezing medium results in a high and stable cell yield in the composition of the present invention when obtained from donor blood with normal or high triglyceride levels. In some embodiments, regardless of the triglyceride level in the donor blood, at least adding an anticoagulant to the incubation medium results in a high and stable cell yield in the composition. In some embodiments, regardless of the triglyceride level in the donor blood, adding an anticoagulant to the freezing medium and the incubation medium results in a high and stable cell yield in the composition.

[0092] In some embodiments, the freezing medium and / or incubation medium and / or wash medium comprises heparin at a concentration of at least 0.1 U / ml, possibly at least 0.3 U / ml, and typically at least 0.5 U / ml. In some embodiments, the freezing medium and / or incubation medium and / or wash medium comprises ACD formula A at a concentration of at least 1% v / v, possibly at least 3% v / v, and typically at least 5% v / v.

[0093] In some embodiments, the cell composition enriched for mononuclear cells undergoes at least one washing step after cell collection and before resuspending in freezing medium and freezing. In some embodiments, the cell composition enriched for mononuclear cells undergoes at least one washing step after freezing and thawing. In some embodiments, the washing step comprises centrifuging the cell composition enriched for mononuclear cells, followed by extracting the supernatant and resuspending in a washing medium.

[0094] In some embodiments, the cell composition enriched in mononuclear cells undergoes at least one washing step between each stage of early apoptotic cell colony generation. In some embodiments, in the washing steps throughout the early apoptotic cell colony generation, an anticoagulant is added to the washing medium. In some embodiments, the cell composition enriched in mononuclear cells undergoes at least one washing step after incubation. In some embodiments, the cell composition enriched in mononuclear cells undergoes at least one washing step using PBS after incubation. In some embodiments, an anticoagulant is not added to the final washing step before the cell product is resuspended in the administration medium. In some embodiments, an anticoagulant is not added to the PBS used in the final washing step before the cell product is resuspended in the administration medium. In certain embodiments, an anticoagulant is not added to the administration medium.

[0095] In some embodiments, the cell concentration during the incubation period is about 5×10 6 cells / ml.

[0096] In some embodiments, after freezing, thawing and incubation, the cell composition enriched in mononuclear cells is suspended in an administration medium to produce a drug population. In some embodiments, the administration medium includes a suitable physiological buffer. Non-limiting examples of suitable physiological buffers are saline solutions, phosphate buffered saline (PBS), Hank's balanced salt solution (HBSS), etc. In some embodiments, the administration medium comprises PBS. In some embodiments, the administration medium comprises a supplement that helps maintain cell viability. In some embodiments, the cell composition enriched in mononuclear cells is filtered before administration. In some embodiments, the cell composition enriched in mononuclear cells is filtered using a filter of at least 200 μm before administration.

[0097] In some embodiments, the mononuclear cell-enriched cell population is resuspended in administration medium such that the final volume of the resulting cell preparation is between 100-1000 ml, possibly between 200-800 ml, and typically between 300-600 ml.

[0098] In some embodiments, cell collection refers to obtaining a cell composition enriched in mononuclear cells. In some embodiments, the washing step performed during the generation of an early apoptotic cell population is performed in a washing medium. In certain embodiments, the washing step before the incubation step to generate an early apoptotic cell population is performed in a washing medium. In some embodiments, the washing medium comprises RPMI 1640 culture medium supplemented with L-glutamine and Hepes. In some embodiments, the washing medium comprises RPMI 1640 culture medium supplemented with 2mM L-glutamine and 10mM Hepes.

[0099] In some embodiments, the wash medium comprises an anticoagulant. In some embodiments, the wash medium comprises an anticoagulant selected from the group consisting of: heparin, ACD formula A, and combinations thereof. In some embodiments, the concentration of the anticoagulant in the wash medium is the same as that in the freezing medium. In some embodiments, the concentration of the anticoagulant in the wash medium is the same as that in the incubation medium. In some embodiments, the anticoagulant used in the wash medium is an ACD formula A containing heparin at a concentration of 10 U / ml.

[0100] In some embodiments, the wash medium comprises heparin. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5U / ml. In some embodiments, the concentration of heparin in the wash medium is between 0.1-2.5U / ml, possibly between 0.3-0.7U / ml, and generally about 0.5U / ml. In certain embodiments, the concentration of heparin in the wash medium is about 0.5U / ml.

[0101] In some embodiments, the wash medium comprises ACD Formula A. In some embodiments, the concentration of ACD Formula A in the wash medium is between 1%-15% v / v. In some embodiments, the concentration of ACD Formula A in the wash medium is between 1%-15% v / v, possibly between 4%-7% v / v, and typically about 5% v / v. In some embodiments, the concentration of ACD Formula A in the wash medium is about 5% v / v.

[0102] In some embodiments, the cell composition enriched for mononuclear cells is thawed several hours before the population is expected to be administered to a subject. In some embodiments, the cell composition enriched for mononuclear cells is thawed at about 33° C.-39° C. In some embodiments, the cell composition enriched for mononuclear cells is thawed for about 30-240 seconds, preferably 40-180 seconds, and most preferably 50-120 seconds.

[0103] In some embodiments, the cell composition of enriched mononuclear cells is thawed at least 10 hours before the expected administration of the colony, and optionally at least 20, 30, 40 or 50 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells is thawed at least 15-24 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells is thawed at least about 24 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells is thawed at least 20 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells is thawed 30 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells is thawed at least 24 hours before the expected administration of the colony. In some embodiments, the cell composition of enriched mononuclear cells undergoes at least one washing step in a washing medium before and / or after thawing.

[0104] In some embodiments, the composition further comprises methylprednisolone. In some embodiments, the concentration of methylprednisolone does not exceed 30 μg / ml.

[0105] In some embodiments, apoptotic cells are used in high doses. In some embodiments, apoptotic cells are used in high concentrations. In some embodiments, human apoptotic polymorphonuclear neutrophils (PMN) are used. In some embodiments, a group of cells is used, 50% of which are apoptotic cells. In some embodiments, apoptotic cells are verified by cytoprep stained with May-Giemsa. In some embodiments, the viability of cells is assessed by trypan blue exclusion. In some embodiments, the apoptotic and necrotic states of cells are confirmed by annexin V / propidium iodide staining and detected with FACS.

[0106] In some embodiments, the apoptotic cells disclosed herein do not comprise necrotic cells. In some embodiments, the apoptotic cells disclosed herein comprise less than 1% necrotic cells. In some embodiments, the apoptotic cells disclosed herein comprise less than 2% necrotic cells. In some embodiments, the apoptotic cells disclosed herein comprise less than 3% necrotic cells. In some embodiments, the apoptotic cells disclosed herein comprise less than 4% necrotic cells. In some embodiments, the apoptotic cells disclosed herein comprise less than 5% necrotic cells.

[0107] In some embodiments, apoptotic cells are prepared from cells obtained from subjects other than the subject to receive the apoptotic cells. In some embodiments, the method as disclosed herein includes additional steps that can be used to overcome allogeneic donor cell rejection, including one or more steps described in U.S. Patent Application Publication 20130156794, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, the method includes a complete or partial lymphocyte depletion step before administering apoptotic cells (in some embodiments, the apoptotic cells are allogeneic apoptotic cells). In some embodiments, lymphocyte depletion is regulated so that it delays host-versus-graft response for a period of time, which is sufficient to allow allogeneic apoptotic cells to control the release of cytokines. In some embodiments, the method includes the step of administering an agent that delays the emigration of allogeneic apoptotic T cells from lymph nodes, such as 2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol (FTY720), 5-[4-phenyl-5-(trifluoromethyl)thiophen-2-yl]-3-[3-(trifluoromethyl)phenyl-1]1,2,4-oxadiazole (SEW2871), 3-(2-(-hexylphenylamino)-2-oxoethylamino)propionic acid (W123), 2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-yl hydrogen phosphate (2-ammonio-4-(2-chloro-4-(3-phenoxyphenylthio)phenyl)-2-(hydroxymethyl)but-yl hydrogen phosphate Phosphate, KRP-203 phosphate) or other agents known in the art can be used as part of the compositions and methods disclosed herein to allow the use of allogeneic apoptotic cells to have efficacy and lack the induction of graft-versus-host disease. In another embodiment, the MHC expression of allogeneic apoptotic T cells is silenced to reduce rejection of allogeneic cells.

[0108] In some embodiments, the method includes producing a population of mononuclear apoptotic cells comprising: a reduced percentage of non-quiescent non-apoptotic viable cells; inhibited cell activation of any viable non-apoptotic cells; or reduced proliferation of any viable non-apoptotic cells; or any combination thereof, the method comprising the steps of obtaining a population of cells enriched for mononuclear cells from peripheral blood; freezing the population of cells enriched for mononuclear cells in a freezing medium comprising an anticoagulant; thawing the population of cells enriched for mononuclear cells; incubating the population of cells enriched for mononuclear cells in an apoptosis-inducing incubation medium comprising methylprednisolone at a final concentration of about 10-100 μg / mL and an anticoagulant; resuspending the population of apoptotic cells in an administration medium; and inactivating the population of enriched mononuclear cells, wherein the inactivation occurs after induction of apoptosis, wherein the method produces a population of mononuclear apoptotic cells comprising: a reduced percentage of non-quiescent non-apoptotic cells; inhibited cell activation of any viable non-apoptotic cells; or reduced proliferation of any viable non-apoptotic cells; or any combination thereof.

[0109] In some embodiments, the method includes the step of irradiating the apoptotic cell population derived from a subject before administering the apoptotic cell population to the same subject (autologous ApoCell; Autocetra). In some embodiments, the method includes the step of irradiating the apoptotic cell population derived from a subject before administering the apoptotic cell population to a recipient (allogeneic ApoCell; Autocetra).

[0110] In some embodiments, cells are irradiated in a manner that will reduce the proliferation and / or activation of residual surviving cells in an apoptotic cell population. In some embodiments, cells are irradiated in a manner that reduces the percentage of non-apoptotic cells that survive in a population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to less than 50% of the population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to less than 40% of the population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to less than 30% of the population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to less than 20% of the population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to less than 10% of the population. In some embodiments, in an inactivated early apoptotic cell population, the percentage of surviving non-apoptotic cells is reduced to 0% of the population.

[0111] In another embodiment, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory and stable properties. In another embodiment, the irradiating step uses UV radiation. In another embodiment, the irradiating step uses gamma radiation. In another embodiment, the apoptotic cells comprise a reduced percentage of viable non-apoptotic cells, a preparation comprising inhibited cell activation of any viable non-apoptotic cells present in the apoptotic cell preparation, or a preparation comprising reduced proliferation of any viable non-apoptotic cells present in the apoptotic cell preparation, or any combination thereof.

[0112] In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+). In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 1%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 2%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 3%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 4%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 5%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 6%. In some embodiments, compared with non-radiated apoptotic cells, radiating apoptotic cells will not increase the population of dead cells (PI+) by more than about 7%. In some embodiments, compared with non-irradiated apoptotic cells, irradiation of apoptotic cells does not increase the number of dead cells (PI+) by more than about 8%. In some embodiments, compared with non-irradiated apoptotic cells, irradiation of apoptotic cells does not increase the number of dead cells (PI+) by more than about 9%. In some embodiments, compared with non-irradiated apoptotic cells, apoptotic cells do not increase the number of dead cells (PI+) by more than about 10%. In some embodiments, compared with non-irradiated apoptotic cells, irradiation of apoptotic cells does not increase the number of dead cells (PI+) by more than about 15%. In some embodiments, compared with non-irradiated apoptotic cells, irradiation of apoptotic cells does not increase the number of dead cells (PI+) by more than about 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0113] In some embodiments, a cell population comprising a reduced proportion or absence of live non-apoptotic cells can provide, in one embodiment, a mononuclear early apoptotic cell population without any live / viable cells. In some embodiments, a cell population comprising a reduced proportion or absence of live non-apoptotic cells can provide, in one embodiment, a mononuclear apoptotic cell population that does not induce GVHD in a recipient.

[0114] In some embodiments, the use of irradiated ApoCells eliminates a possible graft-versus-leukemia effect that may be caused by the use of an apoptotic cell population (including a small fraction of surviving cells), demonstrating that the effects shown herein in the Examples (see Example 3) are derived from apoptotic cells, rather than from a surviving, proliferating population of cells with cell viability present in the apoptotic cell population.

[0115] In another embodiment, the method includes a step of irradiating apoptotic cells derived from donor WBCs before administering to the recipient. In some embodiments, the cells are irradiated in a manner that will avoid proliferation and / or activation of the remaining viable cells in the apoptotic cell population. In another embodiment, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory, stable characteristics. In another embodiment, the irradiation step uses UV radiation. In another embodiment, the irradiation step uses gamma radiation. In another embodiment, the apoptotic cells comprise live non-apoptotic cells with a reduced percentage, a product in which the cell activation of any live non-apoptotic cells present in the apoptotic cell product is suppressed, or a product in which the cell proliferation of any live non-apoptotic cells present in the apoptotic cell product is reduced, or any combination thereof.

[0116] In some embodiments, the apoptotic cells comprise a pooled mononuclear apoptotic cell preparation. In some embodiments, the pooled mononuclear apoptotic cell preparation comprises mononuclear cells in an early apoptotic state, wherein the pooled mononuclear apoptotic cells comprise a reduced percentage of live non-apoptotic cells, a preparation comprising any live non-apoptotic cells in which cell activation is inhibited, or a preparation comprising any live non-apoptotic cells in which proliferation is reduced, or any combination thereof. In another embodiment, the pooled mononuclear apoptotic cells have been irradiated. In another embodiment, the pooled mononuclear apoptotic cell preparation is disclosed herein, and in some embodiments, the preparation is derived from a white blood cell fraction (WBC) obtained from donated blood.

[0117] In some embodiments, the apoptotic cell preparation is irradiated. In another embodiment, the irradiation comprises gamma radiation or UV radiation. In yet another embodiment, the irradiated preparation has a reduced number of non-apoptotic cells compared to a non-irradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of proliferating cells compared to a non-irradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of potentially immunocompetent cells compared to a non-irradiated apoptotic cell population.

[0118] In some embodiments, the pooled blood includes third party blood that is not matched between the donor and recipient (allogeneic).

[0119] Those skilled in the art will appreciate that the term "pooling" may include collecting, preparing and possibly storing blood from more than one donor for subsequent use. This combined blood pool may then be processed to produce a pooled mononuclear apoptotic cell preparation. In another embodiment, the pooled mononuclear apoptotic cell preparation ensures that a readily available supply of mononuclear apoptotic cells is available. In another embodiment, the cells are pooled just prior to the incubation step for inducing apoptosis. In another embodiment, the cells are pooled after the incubation step of the resuspension step. In another embodiment, the cells are pooled just prior to the irradiation step. In another embodiment, the cells are pooled after the irradiation step. In another embodiment, the cells are pooled at any step of the preparation method.

[0120] In some embodiments, the pooled mononuclear apoptotic cell population comprises blood or apoptotic cells pooled at any step of the early apoptotic cell process of preparation. In some embodiments, the cells are allogeneic. In some embodiments, the cells are autologous.

[0121] In some embodiments, the apoptotic cell preparations collected are derived from cells present in between about 2 and 25 units of blood. In another embodiment, the apoptotic cell preparations collected comprise cells present in about 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 5-25, 10-15, 10-20, 10-25, 6-13 or 6-25 units of blood. In another embodiment, the apoptotic cell preparations collected comprise cells present in about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 units of blood. The number of units of blood required also depends on the efficiency of recovering WBCs from the blood. For example, inefficient WBC recovery will result in the need for additional units, while efficient WBC recovery will result in the need for fewer units. In some embodiments, each unit is a bag of blood. In another embodiment, the pooled apoptotic cell preparation comprises cells present in at least 25 units of blood, at least 50 units of blood, or at least 100 units of blood.

[0122] In some embodiments, the blood unit includes a white blood cell (WBC) fraction from a donated blood. In another embodiment, the donation can come from a blood center or a blood bank. In another embodiment, the donation can come from a donor gathered in a hospital when preparing a pooled apoptotic cell product. In another embodiment, a blood unit containing WBC from more than one donor is preserved and maintained in an independent blood bank created for the purpose of the compositions disclosed herein and methods thereof. In another embodiment, a blood bank developed for the purpose of the compositions disclosed herein and methods thereof can provide a blood unit containing WBC from more than one donor and contains a leukocyte apheresis unit.

[0123] In some embodiments, the pooled WBC units are not restricted by HLA matching. Thus, the resulting pooled apoptotic cell preparation comprises a cell population that is not restricted by HLA matching. Thus, in certain embodiments, the pooled mononuclear apoptotic cell preparation comprises allogeneic cells.

[0124] The advantages of pooled mononuclear apoptotic cell preparations derived from pooled WBCs, without the restriction of HLA matching, are the easy availability of WBC sources and the reduced cost of obtaining WBCs.

[0125] In some embodiments, the collected blood includes blood from more than one donor, independent of HLA matching. In another embodiment, the collected blood includes blood from more than one donor, wherein HLA matching with the recipient is considered. For example, wherein 1 HLA allele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6 HLA alleles or 7 HLA alleles have been matched between the donor and the recipient. In another embodiment, more than one donor is partially matched, such as some donors are already HLA matched, wherein 1 HLA allele, 2 HLA alleles, 3 HLA alleles, 4 HLA alleles, 5 HLA alleles, 6 HLA alleles or 7 HLA alleles have been matched between some donors and recipients. Each possibility constitutes an embodiment disclosed herein.

[0126] In certain embodiments, after the following apoptosis induction step (Example 1), some surviving non-apoptotic cells (anti-apoptotic) may remain. In some embodiments, the presence of these surviving non-apoptotic cells is observed before the radiation step. These surviving non-apoptotic cells may be able to proliferate or be activated. In some embodiments, the mononuclear apoptotic cell preparations from more than one donor pool may be activated for the host, for each other, or both.

[0127] In some embodiments, compared with non-irradiated cell products, the irradiated cell products disclosed herein have inhibited cell activation and reduced proliferation. In another embodiment, the radiation includes gamma radiation or UV radiation. In another embodiment, compared with non-irradiated cell products, the irradiated cell products have a reduced number of non-apoptotic cells. In some embodiments, the radiation includes about 10 gray units (Gy) to 60Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 6Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 50Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 40Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 30Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 20Gy. In some embodiments, the radiation includes about 10 gray units (Gy) to 15Gy.

[0128] In another embodiment, the radiation includes about 10 gray units (Gy). In another embodiment, the radiation includes about 15 gray units (Gy). In another embodiment, the radiation includes about 20 gray units (Gy). In another embodiment, the radiation includes about 25 gray units (Gy). In another embodiment, the radiation includes about 30 gray units (Gy). In another embodiment, the radiation includes about 35 gray units (Gy). In another embodiment, the radiation includes about 40 gray units (Gy). In another embodiment, the radiation includes about 45 gray units (Gy). In another embodiment, the radiation includes about 50 gray units (Gy). In another embodiment, the radiation includes about 55 gray units (Gy). In another embodiment, the radiation includes about 60 gray units (Gy). In another embodiment, the irradiated pooled apoptotic cell preparation maintains the same or similar apoptotic distribution, stability and efficacy as the non-irradiated pooled apoptotic cell preparation.

[0129] In some embodiments, the pooled mononuclear apoptotic cell preparations disclosed herein are stable for up to 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 24 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 24 hours. In yet another embodiment, the pooled mononuclear apoptotic cell preparations disclosed herein are stable for up to 36 hours. In yet another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 36 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 36 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations disclosed herein are stable for up to 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for at least 48 hours. In another embodiment, the pooled mononuclear apoptotic cell preparations are stable for more than 48 hours.

[0130] In some embodiments, the method of producing a pooled cell preparation comprising a radiation step retains the early apoptosis, immunomodulation and stability characteristics observed in apoptotic preparations derived from a single matched donor, wherein the cell preparation may not include a radiation step. In another embodiment, the pooled mononuclear apoptotic cell preparation disclosed herein does not induce a graft-versus-host disease (GVHD) response.

[0131] Irradiation of cell products is considered safe in the art. Donated blood is currently subjected to routine irradiation procedures to prevent reactions with WBCs.

[0132] In another embodiment, the percentage of apoptotic cells in the mononuclear apoptotic cell preparations disclosed herein is close to 100%, thereby reducing the proportion of live non-apoptotic cells in the cell preparations. In some embodiments, the percentage of apoptotic cells is at least 40%. In another embodiment, the percentage of apoptotic cells is at least 50%. In another embodiment, the percentage of apoptotic cells is at least 60%. In yet another embodiment, the percentage of apoptotic cells is at least 70%. In yet another embodiment, the percentage of apoptotic cells is at least 80%. In another embodiment, the percentage of apoptotic cells is at least 90%. In yet another embodiment, the percentage of apoptotic cells is at least 99%. Therefore, in one embodiment, a cell preparation comprising a reduced proportion or the absence of live non-apoptotic cells can provide a mononuclear apoptotic cell preparation that does not induce GVHD in the recipient. Each possibility represents an embodiment disclosed herein.

[0133] Alternatively, in another embodiment, by specifically removing the live cell colony, for example, by targeted precipitation, the percentage of live non-apoptotic WBC is reduced. In another embodiment, magnetic beads in conjunction with phosphatidylserine can be used to reduce the percentage of live non-apoptotic cells. In another embodiment, magnetic beads in conjunction with markers on the cell surface of non-apoptotic cells rather than apoptotic cells can be used to reduce the percentage of live non-apoptotic cells. In another embodiment, magnetic beads can be used to select apoptotic cells for further preparation, and the magnetic beads are in conjunction with markers on the cell surface of apoptotic cells rather than non-apoptotic cells. In yet another embodiment, the percentage of live non-apoptotic WBC is reduced by using ultrasound.

[0134] In one embodiment, the apoptotic cells are from pooled third party donors.

[0135] In some embodiments, the collected cell preparations contain at least one cell type selected from the group consisting of: lymphocytes, monocytes and natural killer cells. In another embodiment, the collected cell preparations contain an enriched population of monocytes. In some embodiments, the collected mononuclear cells are cell preparations enriched for mononuclear cells, which contain cell types selected from the group consisting of: lymphocytes, monocytes and natural killer cells. In another embodiment, the cell preparations enriched for mononuclear cells contain no more than 15%, optionally no more than 10%, and usually no more than 5% of polymorphonuclear leukocytes, which are also called granulocytes (i.e., neutrophils, basophils and eosinophils). In another embodiment, the mixed mononuclear cell preparations do not contain granulocytes.

[0136] In another embodiment, the pooled mononuclear cell-enriched cell preparation contains no more than 15%, optionally no more than 10%, and usually no more than 5% of CD15 high-expressing cells. In some embodiments, the pooled apoptotic cell preparation contains less than 15% of CD15 high-expressing cells.

[0137] In some embodiments, the pooled mononuclear cell-enriched cell preparation disclosed herein comprises at least 80% mononuclear cells, at least 85% mononuclear cells, alternatively at least 90% mononuclear cells, or at least 95% mononuclear cells, wherein each possibility is a separate embodiment disclosed herein. According to some embodiments, the pooled mononuclear cell-enriched cell preparation disclosed herein comprises at least 85% mononuclear cells.

[0138] In another embodiment, any pooled cell preparation with at least 80% of the final pooled percentage of mononuclear cells is considered to be a pooled cell preparation enriched for mononuclear cells disclosed herein. Thus, a cell preparation with increased polymorphonuclear cells (PMN) is pooled with a cell preparation with high mononuclear cells to obtain a "pool" with at least 80% mononuclear cells, constituting a preparation disclosed herein. According to some embodiments, mononuclear cells include lymphocytes and monocytes.

[0139] The skilled artisan will appreciate that the term "mononuclear cell" may include leukocytes having one lobed nucleus. In another embodiment, the pooled apoptotic cell preparation disclosed herein comprises less than 5% polymorphonuclear leukocytes.

[0140] In some embodiments, the apoptotic cells are T cells. In another embodiment, the apoptotic cells and CAR T cells are derived from the same pooled third-party donor T cells. In another embodiment, the apoptotic cells are derived from a CAR T cell population.

[0141] Supernatant-apoptotic cell supernatant and apoptotic cell-phagocyte supernatant

[0142] In some embodiments, apoptotic cell supernatants useful for the methods and treatments disclosed herein include apoptotic cell supernatants and apoptotic phagocyte supernatants disclosed herein. In some embodiments, the cells from which the supernatant is collected are autologous to the patient. In some embodiments, the cells from which the supernatant is collected are allogeneic, from a donor. In some embodiments, the cells from which the supernatant is collected are allogeneic, from cells collected from a blood bank.

[0143] In some embodiments, the apoptotic cell supernatant is obtained by a method comprising the steps of: a) providing apoptotic cells, b) culturing the apoptotic cells of step a), and c) separating the cells from the supernatant. In certain embodiments, the apoptotic cells for obtaining the supernatant are prepared by any method disclosed herein. In certain embodiments, the apoptotic cells for obtaining the supernatant are prepared by any method known in the art.

[0144] In some embodiments, the apoptotic cells used to prepare the apoptotic cell supernatants disclosed herein are autologous to the subject being treated. In another embodiment, the apoptotic cells used to prepare the apoptotic cell supernatants disclosed herein are allogeneic to the subject being treated.

[0145] The "apoptotic cells" from which the apoptotic cell supernatant is obtained can be cells of any cell type selected from a subject, or any commercially available cell line, that have undergone a method for inducing apoptosis known to those skilled in the art. The method for inducing apoptosis can be hypoxia, ozone, heat, radiation, chemicals, osmotic pressure, pH changes, X-ray radiation, gamma ray radiation, UV radiation, serum deprivation, corticosteroids, or a combination thereof, or any other method described herein or known in the art. In another embodiment, the method for inducing apoptosis produces apoptotic cells in an early apoptotic state.

[0146] In some embodiments, the apoptotic cells are leukocytes.

[0147] In one embodiment, the apoptotic leukocytes are derived from peripheral blood mononuclear cells (PBMCs). In another embodiment, the leukocytes are from pooled third party donors. In another embodiment, the leukocytes are allogeneic.

[0148] According to some embodiments, by selecting non-adhesive leukocytes and inducing apoptosis, a cell culture step is subsequently performed in culture medium to provide apoptotic cells. "Leukocytes" for preparing apoptotic cell-phagocyte supernatant can be derived from any pedigree or sub-pedigree of nucleated cells of immune system and / or hematopoietic system, including but not limited to dendritic cells, macrophages, mast cells, basophils, hematopoietic stem cells, bone marrow cells, natural killer cells, etc. According to application and purpose, desired leukocyte pedigree, etc., according to any of various commonly used methods, leukocytes can be derived or obtained in any of various suitable ways, from any of various suitable anatomical compartments. In some embodiments, the source leukocytes are primary leukocytes. In another embodiment, the source leukocytes are primary peripheral blood leukocytes.

[0149] Primary lymphocytes and monocytes can be conveniently derived from peripheral blood. Peripheral blood leukocytes contain 70%-95% lymphocytes and 5%-25% monocytes.

[0150] Methods for obtaining specific types of source leukocytes from blood are routinely implemented. Obtaining source lymphocytes and / or monocytes can be achieved by, for example, collecting blood in the presence of an anticoagulant such as heparin or citrate. The collected blood is then centrifuged on a Ficoll cushion to separate lymphocytes and monocytes at the gradient interface, and neutrophils and erythrocytes in the precipitate.

[0151] Leukocytes can be separated from each other based on their specific surface markers by standard immunomagnetic selection or immunofluorescence flow cytometry techniques or by centrifugal elutriation. For example, monocytes can be selected as CD14 + The fraction was selected, and T lymphocytes were used as CD3 + The fraction was selected, and B lymphocytes could be used as CD19 + The fraction was selected and macrophages were identified as CD206 + The fractions are selected.

[0152] Lymphocytes and monocytes can be separated from each other by placing these cells under substrate adhesion conditions, such as by static culture in tissue culture treated culture vessels, which results in the selective adhesion of monocytes but not lymphocytes to the cell adhesion substrate.

[0153] Leukocytes can also be obtained from peripheral blood mononuclear cells (PBMCs), which can be isolated as described herein.

[0154] One of ordinary skill in the art will possess the necessary expertise to appropriately culture primary leukocytes to produce the desired quantities of cultured source leukocytes disclosed herein, and ample guidance for practicing such culture methods is available in the literature in the art.

[0155] Those of ordinary skill in the art will also have the necessary expertise to establish, purchase or otherwise obtain suitable established leukocyte cell lines from which apoptotic leukocytes are derived. Suitable leukocyte cell lines can be obtained from commercial suppliers, such as the American Type Culture Collection (ATCC). It will be apparent to those skilled in the art that source leukocytes should not be obtained by techniques that would significantly interfere with their ability to produce apoptotic leukocytes.

[0156] In another embodiment, apoptotic cells can be apoptotic lymphocytes. The apoptosis of lymphocytes such as primary lymphocytes can be induced by serum deprivation, corticosteroids or radiation treatment of primary lymphocytes. In another embodiment, the apoptosis of primary lymphocytes induced by treatment with corticosteroids is achieved by treating primary lymphocytes with dexamethasone. In another embodiment, the concentration of dexamethasone is about 1 micromole / liter. In another embodiment, the apoptosis of primary lymphocytes induced by radiation is achieved by treating primary lymphocytes with gamma-radiation. In another embodiment, the dosage is about 66rad. Such treatment results in the generation of apoptotic lymphocytes suitable for co-culturing steps with phagocytes.

[0157] In another embodiment, the apoptotic cells can be apoptotic monocytes, such as primary monocytes. To generate apoptotic monocytes, the monocytes are subjected to in vitro conditions of substrate / surface adhesion under serum deprivation. Such treatment results in the generation of non-proinflammatory apoptotic monocytes suitable for co-culturing steps with phagocytes.

[0158] In other embodiments, the apoptotic cell can be any apoptotic cell described herein, including allogeneic apoptotic cells, third party apoptotic cells, and pools of apoptotic cells.

[0159] In other embodiments, apoptotic cell supernatant can be obtained by co-culturing apoptotic cells with other cells. In some embodiments, apoptotic cell supernatant can be obtained by co-culturing apoptotic cells with leukocytes. In some embodiments, apoptotic cell supernatant can be obtained by co-culturing apoptotic cells with dendritic cells. In some embodiments, apoptotic cell supernatant can be obtained by co-culturing apoptotic cells with macrophages. Macrophages can be produced in different ways, including M1 macrophages and M2 macrophages.

[0160] In some embodiments, the apoptotic cell supernatant can be obtained by culturing leukocytes that have taken up apoptotic cells or fragments thereof. In some embodiments, the apoptotic cell supernatant can be obtained by culturing dendritic cells that have taken up apoptotic cells or fragments thereof. In some embodiments, the apoptotic cell supernatant can be obtained by culturing macrophages that have taken up apoptotic cells or fragments thereof.

[0161] In some embodiments, the apoptotic cell supernatant is an apoptotic cell supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing other cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant.

[0162] Those skilled in the art will understand that, as used herein in certain embodiments, the term "supernatant" may be used interchangeably with "apoptotic cell supernatant," "apoptotic supernatant," "apoptotic-phagocyte supernatant," "apoptotic-leukocyte supernatant," "apoptotic-macrophage supernatant," "apoptotic-dendritic supernatant," etc., although the source and preparation method of the supernatant may be different, the above terms have the same meaning and quality of affecting cytokine release or treating osteoarthritis or vanishing bone disease or a combination thereof.

[0163] In some embodiments, the other cells co-cultured with the apoptotic cells are leukocytes. In some embodiments, the leukocytes can be phagocytic cells, such as macrophages, monocytes, or dendritic cells. In some embodiments, the leukocytes can be B cells, T cells, or natural killer cells (NK cells).

[0164] In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing leukocytes, c) optionally washing cells from steps a) and b), d) co-culturing cells of steps a) and b), and optionally e) separating cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing phagocytes, c) optionally washing cells from steps a) and b), d) co-culturing cells of steps a) and b), and optionally e) separating cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing macrophages, c) optionally washing cells from steps a) and b), d) co-culturing cells of steps a) and b), and optionally e) separating cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing dendritic cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing monocytes, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing B cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing T cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant. In certain embodiments, the apoptotic cell supernatant is an apoptotic cell-leukocyte supernatant obtained by a method comprising the steps of: a) providing apoptotic cells, b) providing natural killer (NK) cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant.

[0165] In some embodiments of the methods of obtaining a cell supernatant, apoptotic cells are first cultured with leukocytes so that the leukocytes will take up the apoptotic cells or portions thereof.

[0166] Thus, in some embodiments, the methods and treatments disclosed herein use apoptotic cell-phagocytic supernatant or a composition thereof, as described in WO 2014 / 106666, which is incorporated herein by reference in its entirety. In another embodiment, the apoptotic cell-phagocytic supernatant or a composition thereof for use in the methods disclosed herein is produced in any manner known in the art.

[0167] In some embodiments, the apoptotic cell-phagocyte supernatant is obtained from a co-culture of phagocytes and apoptotic cells.

[0168] In some embodiments, the apoptotic cell-phagocyte supernatant is obtained by a method comprising the steps of: a) providing phagocytes, b) providing apoptotic cells, c) optionally washing the cells from steps a) and b), d) co-culturing the cells of steps a) and b), and optionally e) separating the cells from the supernatant.

[0169] The term "phagocyte" refers to cells that protect the body by taking in (phagocytizing) harmful foreign particles, bacteria, and dead or dying cells. Phagocytic cells include, for example, cells called neutrophils, monocytes, macrophages, dendritic cells, and mast T cells, preferably dendritic cells and monocytes / macrophages. Phagocytic cells can be dendritic cells (CD4+HLA-DR+ lineage-BDCA1 / BDCA3+), macrophages (CD14+CD206+HLA-DR+), or derived from monocytes (CD14+). The techniques for distinguishing these different phagocytes are known to those skilled in the art.

[0170] In one embodiment, monocytes are obtained by a plastic adherence step. The monocytes can be distinguished from B cells and T cells using the marker CD14+, while unwanted B cells express CD19+ and T cells express CD3+. After macrophage colony stimulating factor (M-CSF) induced maturation, in some embodiments, the obtained macrophages are positive for markers CD14+, CD206+, HLA-DR+.

[0171] In one embodiment, the phagocytic cells are derived from peripheral blood mononuclear cells (PBMC).

[0172] The phagocytic cells may be provided by any method known in the art for obtaining phagocytic cells. In some embodiments, phagocytic cells such as macrophages or dendritic cells may be isolated directly from a subject, or obtained from precursor cells through a maturation step.

[0173] In some embodiments, macrophages can be isolated directly from the peritoneal cavity of a subject and cultured in complete RRPMI medium. Macrophages can also be isolated from the spleen.

[0174] Phagocytic cells can also be obtained from peripheral blood mononuclear cells. In such embodiments, when macrophage colony stimulating factor (M-CSF) is added to the cell culture medium, the cultured monocytes differentiate into monocyte-derived macrophages.

[0175] For example, phagocytes can be derived from peripheral blood mononuclear cells (PBMC). For example, PBMC can be separated from a cell purification bag (cytapheresis bag) from an individual by Ficoll gradient centrifugation, and plated with a cell adhesion step for 90 minutes in a complete RPMI culture medium (10% FBS, 1% penicillin / streptomycin). Non-adhesive T cells are removed by a plastic adhesion step, and adherent T cells are cultured in a complete RPMI environment supplemented with recombinant human M-CSF. After the culture period, monocyte-derived macrophages are obtained.

[0176] Phagocytic cells can be selected by cell adhesion step.Described " cell adhesion step " refers to, select phagocytic cells or can mature into the cell of phagocytic cells by culture conditions, and described culture conditions allow the cultured cells to adhere to the surface, i.e. cell adhesion surface (such as tissue culture dish, matrix, capsule or bag with suitable type nylon or plastic).Technician will understand that term " cell adhesion surface " can include hydrophilic and negatively charged, and can obtain in any one of various ways known in the art.In another embodiment, by using for example corona discharge or gas plasma to modify polystyrene surface.These processes produce high energy oxygen ions, and these oxygen ions are grafted on the surface polystyrene chain, make the surface become hydrophilic and negatively charged.From various commercial suppliers (such as, Corning, Perkin-Elmer, FisherScientific, Evergreen Scientific, Nunc etc.), all can obtain the culture vessel designed to promote cell adhesion.

[0177] B cell, T cell and NK cell can be provided by any method known in the art for obtaining such cell.In some embodiments, B cell, T cell or NK cell can be directly separated from subject, or derived from precursor cell by maturation step.In another embodiment, B, T or NK cell can be from B, T or NK cell line.One of ordinary skill in the art will have the necessary expertise of establishing, purchasing or otherwise obtaining the B cell, T cell and NK cell line of suitable establishment.Suitable cell line can be obtained from commercial supplier, such as American Type Culture Collection (ATCC).

[0178] In one embodiment, prior to the co-culturing step d), said apoptotic cells and said leukocytes, such as phagocytes, B cells, T cells or NK cells, are cultured separately.

[0179] Cell maturation of phagocytes occurs during cell culture, for example due to the addition of maturation factors to the culture medium. In one embodiment, the maturation factor is M-CSF, which can be used, for example, to obtain monocyte-derived macrophages.

[0180] The culturing step for maturation or selection of phagocytes may take from several hours to several days. In another embodiment, the pre-mature phagocytes are cultured in a suitable culture medium for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours.

[0181] Culture medium for phagocytic cells is known to those skilled in the art and may be, for example, but not limited to, RPMI, DMEM, X-vivo and Ultraculture milieus.

[0182] In one embodiment, the co-culture of apoptotic cells and phagocytes is performed in a physiological solution.

[0183] Prior to this "co-cultivation", the cells may be subjected to a washing step. In some embodiments, prior to the co-cultivation step, leukocytes (e.g., phagocytes) and apoptotic cells are washed. In another embodiment, the cells are washed with PBS.

[0184] In the co-culture process, leukocytes (e.g., phagocytic cells such as macrophages, monocytes or phagocytes, or B, T or NK cells) and apoptotic cells can be mixed in a ratio of 10: 1, 9: 1; 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1 or 1: 1, or (leukocytes: apoptotic cells) 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9 or 1: 10. In one example, the ratio of leukocytes to apoptotic cells is 1: 5.

[0185] The co-culture of cells may take from several hours to several days. In some embodiments, the apoptotic cells are cultured for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours. Those skilled in the art can assess the optimal time for co-culture by measuring the presence of anti-inflammatory compounds, the amount of leukocyte survival, and the amount of apoptotic cells that have not been eliminated so far. Due to the disappearance of apoptotic cells, the elimination of apoptotic cells by phagocytes can be observed by optical microscopy.

[0186] In some embodiments, culturing of apoptotic cells, e.g., co-culturing with leukocytes (e.g., phagocytic cells such as macrophages, monocytes or phagocytes, or B, T or NK cells), is carried out in culture medium and / or in a physiological solution compatible with administration, e.g., injection, to a subject.

[0187] A skilled artisan will appreciate that a "physiological solution" may include a solution that does not cause death of leukocytes during the culture time. In some embodiments, a physiological solution does not cause death within 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, 50 hours, 52 hours. In other embodiments, 48 ​​hours or 30 hours.

[0188] In some embodiments, leukocytes (e.g., phagocytic cells such as macrophages, monocytes or phagocytes, or B, T or NK cells) and apoptotic cells are incubated in a physiological solution for at least 30 minutes. This period of culture allows phagocytosis to begin and secretion of cytokines and other beneficial substances.

[0189] In one embodiment, such physiological solutions do not inhibit the elimination of apoptotic leukocytes by leukocyte-derived macrophages.

[0190] At the end of the cultivation or co-cultivation step, the supernatant is optionally separated from the cultured apoptotic cells or co-cultivated cells. Techniques for separating cells from supernatants are known in the art. For example, the supernatant can be collected and / or filtered and / or centrifuged to remove cells and debris. For example, the supernatant can be centrifuged at room temperature for 15 minutes at 3000 rpm to separate it from the cells.

[0191] The supernatant may be "inactivated" prior to use, for example by irradiation. Thus, the method for preparing apoptotic cell supernatant may include an optional additional irradiation step f). The "irradiation" step may be considered a sterilization method using X-ray radiation (25-45 Gy) at a sufficient rate to kill microorganisms, as is routinely done for the inactivation of blood products.

[0192] Irradiated supernatants are considered safe in the art. Currently donated blood is routinely irradiated to prevent reactions with WBCs.

[0193] In one embodiment, the apoptotic cell supernatant is formulated into a pharmaceutical composition suitable for administration to a subject, as described in detail herein.

[0194] In some embodiments, the final product is stored at +4° C. In another embodiment, the final product is used within the next 48 hours.

[0195] In some embodiments, apoptotic cell supernatant, such as apoptotic cell-phagocyte supernatant, or a pharmaceutical composition comprising the supernatant, can be lyophilized, such as stored at -80°C.

[0196] In a specific embodiment, as described in Example 1 of WO 2014 / 106666, thymocytes can be used as apoptotic cells to prepare apoptotic cell-phagocytic cell supernatant. After isolation, thymocytes are irradiated (e.g., with 35X-Gray) and cultured in complete DMEM medium for, for example, 6 hours to allow apoptosis to occur. In parallel, macrophages are isolated from the peritoneal cavity, washed and cultured in complete RPMI (10% FBS, Peni-Strepto, EAA, Hepes, NaP and 2-mercaptoethanol). The macrophages and apoptotic cells are then washed and co-cultured in phenol-free X-vivo medium for another 48 hours at a ratio of 1 / 5 macrophages / apoptotic cells. The supernatant is then collected, centrifuged to remove debris, and can be frozen or freeze-dried for storage. The enrichment of macrophages can be confirmed by positive staining for F4 / 80 by FACS. Cell apoptosis can be confirmed by positive staining for annexin-V and exclusion of 7AAD by FACS.

[0197] In one embodiment, the apoptotic cell supernatant is enriched in TGF-β levels in both active and latent forms of TGF-β compared to supernatants obtained from macrophages or apoptotic cells cultured alone. In one embodiment, IL-10 levels are also increased compared to macrophages cultured alone, and significantly increased compared to apoptotic cells cultured alone. In another embodiment, inflammatory cytokines such as IL-6 are not detectable, and IL-1β and TNF are not detectable or at very low levels.

[0198] In embodiments, apoptotic cell supernatants have increased levels of IL-1ra, TIMP-1, CXCL1 / KC, and CCL2 / JE / MCP1, in addition to TGF-β and IL-10, when compared to supernatants from macrophages cultured alone or apoptotic cells cultured alone, which may involve the tolerogenic effect of the supernatant in controlling inflammation.

[0199] In another specific embodiment, as described in Example 3 of WO 2014 / 106666, human apoptotic cell-phagocyte supernatant can be made from a co-culture of macrophages derived from peripheral blood mononuclear cells (PBMC) and apoptotic PBMCs. Therefore, PBMCs are isolated from cell purification bags from healthy volunteers by, for example, Ficoll gradient centrifugation. The PBMCs are then plated in complete RPMI medium (10% FBS, 1% penicillin / streptomycin) for 90 minutes. Then, non-adherent T cells are removed and apoptotic using, for example, 35Gy doses of X-ray radiation, and cultured in a complete RPMI environment for 4 days (including cell washing after the first 48 hours of culture) to allow apoptosis to occur. In parallel, adherent T cells are cultured in a complete RPMI environment supplemented with 50 μg / mL recombinant human M-CSF for 4 days, including cell washing after the first 48 hours. At the end of the 4-day culture period, the monocyte-derived macrophages and apoptotic cells are washed and cultured together again for 48 hours in X-vivo medium at a ratio of 1 macrophage to 5 apoptotic cells. The supernatant of the latter culture is then collected, centrifuged to remove cells and debris, and can be frozen or lyophilized for storage and subsequent use.

[0200] In one embodiment, human apoptotic cell-phagocyte supernatant can be obtained from peripheral blood mononuclear cells (PBMC) within 6 days as described in WO 2014 / 106666. PBMC-derived macrophages are obtained by adding M-CSF to the culture for 4 days, and then the PBMC-derived macrophages are co-cultured with apoptotic cells for another 2 days, corresponding to non-adherent PBMC cells isolated on day 0.

[0201] In one embodiment, as described in WO 2014 / 106666, standardized human apoptotic cell-phagocytic supernatant can be obtained independently of the donor or PBMC source (cell purification or buffy coat). The plastic adhesion step is sufficient to obtain a significant starting population enriched in monocytes (after adhesion to plastic culture dishes, the proportion of CD14+ cells is 20%-93%). In addition, such adherent T cells show very low presence of B cells and T cells (1.0% CD19+B cells and 12.8% CD3+T cells). After culturing adherent T cells in the presence of M-CSF for 4 days, the proportion of monocyte-derived macrophages increased significantly from 0.1% of CD14+CD206+HLA-DR+ macrophages to 77.7%. At this point, monocyte-derived macrophages can be co-cultured with apoptotic non-adherent PBMC cells (47.6% apoptosis, as shown by annexin V staining and 7AAD exclusion) to produce apoptotic cell-phagocytic supernatant within 48 hours.

[0202] In embodiments, the collected apoptotic cell-phagocyte supernatant contains significantly more latent TGF than in the incubation supernatant of monocyte-derived macrophages alone or monocyte-derived macrophages treated with inflammatory conditions (+LPS), and contains only trace or low levels of inflammatory cytokines, such as IL-1β or TNF.

[0203] In another embodiment, apoptotic supernatants are prepared using early apoptotic cells and monocytes as described in Example 3 below.

[0204] In some embodiments, the composition comprising apoptotic cell supernatant further comprises an anticoagulant. In some embodiments, the anticoagulant is selected from the group consisting of heparin, acid citrate dextran (ACD) formula A, and combinations thereof.

[0205] In another embodiment, an anticoagulant is added during the manufacture of the apoptotic cells used. In another embodiment, the anticoagulant added is selected from the group comprising ACD and heparin or any combination thereof. In another embodiment, the concentration of ACD is 1%. In another embodiment, the concentration of ACD is 2%. In another embodiment, the concentration of ACD is 3%. In another embodiment, the concentration of ACD is 4%. In another embodiment, the concentration of ACD is 5%. In another embodiment, the concentration of ACD is 6%. In another embodiment, the concentration of ACD is 7%. In another embodiment, the concentration of ACD is 8%. In another embodiment, the concentration of ACD is 9%. In another embodiment, the concentration of ACD is 10%. In another embodiment, the concentration of ACD is between about 1%-10%. In another embodiment, the concentration of ACD is between about 2%-8%. In another embodiment, the concentration of ACD is between about 3%-7%. In another embodiment, the concentration of ACD is between about 1%-5%. In another embodiment, the concentration of ACD is between about 5%-10%. In another embodiment, the final concentration of heparin is 0.5U / ml. In another embodiment, the final concentration of heparin is about 0.1U / ml-1.0U / ml. In another embodiment, the final concentration of heparin is about 0.2U / ml-0.9U / ml. In another embodiment, the final concentration of heparin is about 0.3U / ml-0.7U / ml. In another embodiment, the final concentration of heparin is about 0.1U / ml-0.5U / ml. In another embodiment, the final concentration of heparin is about 0.5U / ml-1.0U / ml. In another embodiment, the final concentration of heparin is about 0.01U / ml-1.0U / ml. In another embodiment, the final concentration of heparin is 0.1U / ml. In another embodiment, the final concentration of heparin is 0.2U / ml. In another embodiment, the final concentration of heparin is 0.3U / ml. In another embodiment, the final concentration of heparin is 0.4U / ml. In another embodiment, the final concentration of heparin is 0.5U / ml. In another embodiment, the final concentration of heparin is 0.6U / ml. In another embodiment, the final concentration of heparin is 0.7U / ml. In another embodiment, the final concentration of heparin is 0.8U / ml. In another embodiment, the final concentration of heparin is 0.9U / ml. In another embodiment, the final concentration of heparin is 1.0U / ml. In another embodiment, the concentration of ACD is 5% and the final concentration of heparin is 0.5U / ml.

[0206] In some embodiments, the composition comprising apoptotic cell supernatant further comprises methylprednisolone. In some embodiments, the concentration of methylprednisolone does not exceed 30 μg / ml.

[0207] Apoptotic cell administration

[0208] Surprisingly, apoptotic cells reduce the production of pro-inflammatory and anti-inflammatory cytokines / chemokines. In some embodiments, administration of apoptotic cells reduces the production of pro-inflammatory cytokines / chemokines, including but not limited to IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα, alone or in combination. In one embodiment, apoptotic cells affect the expression level of cytokines in synovial fluid. In one embodiment, apoptotic cells affect the expression level of cytokines in joint fluid.

[0209] In some embodiments, apoptotic cells are administered to inhibit one or more proinflammatory and anti-inflammatory cytokines. In some embodiments, apoptotic cells are administered to inhibit one or more proinflammatory cytokines. In some embodiments, apoptotic cells are administered to inhibit one or more anti-inflammatory cytokines. In some embodiments, proinflammatory and anti-inflammatory cytokines include IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα or any combination thereof. In another embodiment, apoptotic cells are administered to promote the secretion of one or more anti-inflammatory cytokines. In some embodiments, at least one cytokine or chemokine with abnormal expression or content is downregulated by administering apoptotic cells. In some embodiments, each cytokine or chemokine with abnormal expression or content is downregulated by administering apoptotic cells.

[0210] In some embodiments, at least one anti-inflammatory cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cells. In some embodiments, at least one pro-inflammatory cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cells. In some embodiments, a combination of pro-inflammatory cytokines and anti-inflammatory cytokines or chemokines with abnormal expression or content is down-regulated by administering apoptotic cells. In some embodiments, any one or any combination of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα with abnormal expression or content is down-regulated by administering apoptotic cells, for example, in the synovial fluid or joint fluid of the joint area. In some embodiments, apoptotic cells are administered to downregulate any one or any combination of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9, or TNFα with abnormal expression or content, for example in the area of ​​vanishing bone disease. (See Example 4)

[0211] In some embodiments, about 100×10 6 -210×10 6 In some embodiments, about 140×10 6 -210×10 6 In some embodiments, about 100×10 6 -140×10 6 In some embodiments, about 10-100×10 6 The dose of apoptotic cells.

[0212] In some embodiments, about 1×10 6 -×10 9 In some embodiments, about 10×10 6 -×10 9 In some embodiments, administration of about 1×10 6 -×10 8 In some embodiments, administration of about 1×10 7 -×10 8 In some embodiments, administration of about 1×10 6 -×10 7In some embodiments, administration of about 1×10 7 -×10 9 In some embodiments, administration of about 1×10 8 -×10 9 The dose of apoptotic cells.

[0213] In some embodiments, about 20×10 6 In some embodiments, about 30×10 6 In some embodiments, about 40×10 6 In some embodiments, about 50×10 6 In some embodiments, 60×10 6 In some embodiments, about 60×10 6 In some embodiments, about 70×10 6 In some embodiments, about 80×10 6 In some embodiments, about 90×10 6 In some embodiments, about 1-15×10 7 In some embodiments, about 10×10 7 In some embodiments, about 11×10 7 In some embodiments, about 12×10 7 In some embodiments, about 13×10 7 In some embodiments, about 14×10 7 In some embodiments, about 15×10 7 The dose of apoptotic cells.

[0214] In some embodiments, administration of 10×10 6 In another embodiment, 10×10 7 In another embodiment, 10×10 8 In another embodiment, 10×10 9 In another embodiment, 10×10 10In another embodiment, 10×10 11 In another embodiment, 10×10 12 In another embodiment, 10×10 5 In another embodiment, 10×10 4 In another embodiment, 10×10 3 In another embodiment, 10×10 2 The dose of apoptotic cells.

[0215] In some embodiments, a high dose of apoptotic cells is administered. In some embodiments, 35×10 6 In another embodiment, 210×10 6 In another embodiment, 70×10 6 In another embodiment, 140×10 6 In another embodiment, 35-210×10 6 The dose of apoptotic cells.

[0216] In some embodiments, a single dose of apoptotic cells is administered. In some embodiments, multiple doses of apoptotic cells are administered. In some embodiments, 2 doses of apoptotic cells are administered. In some embodiments, 3 doses of apoptotic cells are administered. In some embodiments, 4 doses of apoptotic cells are administered. In some embodiments, 5 doses of apoptotic cells are administered. In some embodiments, 6 doses of apoptotic cells are administered. In some embodiments, 7 doses of apoptotic cells are administered. In some embodiments, 8 doses of apoptotic cells are administered. In some embodiments, 9 doses of apoptotic cells are administered. In some embodiments, more than 9 doses of apoptotic cells are administered. In some embodiments, multiple doses of apoptotic cells are administered. In some embodiments, multiple doses of apoptotic cells are administered during treatment of a subject in need thereof.

[0217] In some embodiments, each dose described herein can be an apoptotic cell dose ± about 5%, 10%, 15%, 20% or 25%. In some embodiments, the dose of apoptotic cells is an apoptotic cell dose ± about 5%. In some embodiments, the dose of apoptotic cells is an apoptotic cell dose ± about 10%. In some embodiments, the dose of apoptotic cells is an apoptotic cell dose ± about 15%. In some embodiments, the dose of apoptotic cells is an apoptotic cell dose ± about 20%. In some embodiments, the dose of apoptotic cells is an apoptotic cell dose ± about 25%. In some embodiments, the dose of apoptotic cells is between an apoptotic cell dose ± about 5%-25%. For example, but not limited to about 100×10 6 -210×10 6 ±20% of the dose, or about 100 × 10 6 ± about 20% of the dose, or 120 × 10 6 ± about 20% of the dose, or 140 × 10 6 ± about 20% of the dose, or 160 × 10 6 ± about 20% of the dose, or 180 × 10 6 ± about 20% of the dose, or 200 × 10 6 ± about 20% of the dose, etc. Similar embodiments include a dose of ± 5%, ± 10%, ± 15%, ± 20% or ± 25%.

[0218] In some embodiments, the dose of apoptotic cells is administered daily. In some embodiments, the dose of apoptotic cells is administered weekly. In some embodiments, the dose of apoptotic cells is administered every half week (twice a week). In some embodiments, the dose of apoptotic cells is administered bi-weekly (every two weeks). In some embodiments, the dose of apoptotic cells is administered monthly. In some embodiments, the dose of apoptotic cells is administered with an irregular regimen, such as daily administration within a given time period, followed by administration every half week, or weekly, or biweekly, or monthly, or a combination thereof.

[0219] In some embodiments, apoptotic cells can be administered by any method known in the art for applying cells directly to a desired area, including but not limited to injection and infusion. In some embodiments, administration comprises injection and / or infusion directly into a joint. In some embodiments, administration comprises injection and / or infusion adjacent to a joint. In some embodiments, administration comprises injection and / or infusion directly at the site of vanishing bone disease. In some embodiments, administration comprises injection and / or infusion near the site of vanishing bone disease. Apoptotic cells for injection can be in the form of a pharmaceutical composition formulated as a sterile injectable solution.

[0220] In some embodiments, apoptotic cells can be administered by topical administration, wherein the cells or pharmaceutical compositions comprising apoptotic cells are applied directly to the area of ​​need. In some embodiments, administration comprises topical application directly to the joint site. In some embodiments, administration comprises topical application adjacent to the joint. In some embodiments, administration comprises topical application directly to the site of vanishing bone disease. In some embodiments, administration comprises topical application adjacent to the site of vanishing bone disease. Apoptotic cells for topical application can be in the form of a pharmaceutical composition formulated as a topical ointment, cream, oil, patch, or skin patch. In some embodiments, apoptotic cells can be administered by infiltrating cartilage in the area of ​​need, wherein the cells or pharmaceutical compositions comprising apoptotic cells are directly targeted to the area of ​​need. In some embodiments, administration comprises infiltrating a joint with apoptotic cells or their compositions. In some embodiments, administration comprises infiltrating tissue adjacent to the joint with apoptotic cells or their compositions. In some embodiments, administration comprises infiltrating an area of ​​the vanishing bone disease site with apoptotic cells or their compositions. In some embodiments, administration comprises infiltrating tissue adjacent to the site of vanishing bone disease with apoptotic cells or their compositions.

[0221] In some embodiments, the apoptotic cells are applied for topical use.

[0222] Apoptotic cell supernatant administration

[0223] In some embodiments, apoptotic cell supernatants reduce the production of proinflammatory and / or anti-inflammatory cytokines / chemokines. In some embodiments, apoptotic cell supernatants are administered to reduce the production of proinflammatory cytokines / chemokines, including but not limited to IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα, alone or in combination. In one embodiment, apoptotic cell supernatants affect the expression level of cytokines in synovial fluid. In one embodiment, apoptotic cell supernatants affect the expression level of cytokines in joint fluid.

[0224] In some embodiments, apoptotic cell supernatant is used to inhibit one or more proinflammatory and anti-inflammatory cytokines. In some embodiments, apoptotic cell supernatant is used to inhibit one or more proinflammatory cytokines. In some embodiments, apoptotic cell supernatant is used to inhibit one or more anti-inflammatory cytokines. In some embodiments, apoptotic cell supernatant is used to inhibit one or more anti-inflammatory cytokines. In some embodiments, proinflammatory and anti-inflammatory cytokines include IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα or any combination thereof. In another embodiment, apoptotic cell supernatant is used to promote the secretion of one or more anti-inflammatory cytokines. In some embodiments, at least one cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cell supernatant. In some embodiments, each cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cell supernatant.

[0225] In some embodiments, at least one anti-inflammatory cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cell supernatant. In some embodiments, at least one pro-inflammatory cytokine or chemokine with abnormal expression or content is down-regulated by administering apoptotic cell supernatant. In some embodiments, a combination of pro-inflammatory cytokines and anti-inflammatory cytokines or chemokines with abnormal expression or content is down-regulated by administering apoptotic cell supernatant. In some embodiments, IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα with abnormal expression or content is down-regulated by administering apoptotic cell supernatant, or any combination thereof, for example, in the synovial fluid or joint fluid of the joint area. In some embodiments, apoptotic cell supernatant is administered to downregulate any one or any combination of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9, or TNFα with aberrant expression or levels, for example in an area of ​​vanishing bone disease.

[0226] In some embodiments, apoptotic supernatant or a composition comprising apoptotic supernatant can be prepared as a cell culture medium derived from about 14×10 9The total dose or aliquot of the apoptotic cell supernatant of the co-culture of CD45+ cells is used, which is equivalent to about 200 million cells per kilogram of body weight (for a 70kg subject). In some embodiments, such a total dose is administered as a supernatant derived from a unit dose of about 100 million cells per kilogram of body weight, and / or is administered at weekly intervals in a unit dose. According to certain embodiments, a suitable total dose includes a total dose of a supernatant derived from about 10 million to about 4 billion cells per kilogram of body weight. In another embodiment, the supernatant is derived from about 40 million to about 1 billion cells per kilogram of body weight. In yet another embodiment, the supernatant is derived from about 80 million to about 500 million cells per kilogram of body weight. In yet another embodiment, the supernatant is derived from about 160 million to about 250 million cells per kilogram of body weight. According to this embodiment, a suitable unit dose includes a unit dose supernatant derived from about 4 million to about 400 million cells per kilogram of body weight. In another embodiment, the supernatant is derived from about 8 million to about 200 million cells per kilogram of body weight. In another embodiment, the supernatant is derived from about 16 million to about 100 million cells per kilogram of body weight. In yet another embodiment, the supernatant is derived from about 32 million to about 50 million cells per kilogram of body weight.

[0227] In another embodiment, administration of a 6 In another embodiment, administration of apoptotic cell supernatant derived from 10×10 7 In another embodiment, administration of apoptotic cells derived from 10×10 8 In another embodiment, administration of apoptotic cells derived from 10×10 9 In another embodiment, administration of apoptotic cells derived from 10×10 10 In another embodiment, administration of apoptotic cells derived from 10×10 11 In another embodiment, administration of apoptotic cells derived from 10×10 12 In another embodiment, administration of apoptotic cells derived from 10×10 5 In another embodiment, administration of apoptotic cells derived from 10×10 4 In another embodiment, administration of apoptotic cells derived from 10×10 3 In another embodiment, administration of apoptotic cells derived from 10×10 2 The dose of apoptotic cells.

[0228] In some embodiments, administration of a protein derived from 35×10 6In another embodiment, administration of apoptotic cell supernatant derived from 210×10 6 In another embodiment, administration of apoptotic cells derived from 70×10 6 In another embodiment, administration of apoptotic cells derived from 140×10 6 In another embodiment, administration of apoptotic cells derived from 35-210×10 6 The dose of apoptotic cells.

[0229] In some embodiments, apoptotic cell supernatants or compositions comprising the apoptotic cell supernatants may be administered.

[0230] In some embodiments, apoptotic cell supernatants, such as apoptotic cell-phagocytic cell supernatants, reduce the production of cytokines associated with cytokine storm, such as IL-6 (see Example 3 below).

[0231] In some embodiments, apoptotic cell supernatants, such as apoptotic cell-phagocyte supernatants, affect cytokine expression levels in macrophages and DCs, but do not affect cytokine expression levels in T cells themselves.

[0232] In another embodiment, apoptotic cell supernatants trigger T cell death, but not through changes in cytokine expression levels.

[0233] In another embodiment, apoptotic cell supernatants, such as apoptotic cell-phagocyte supernatants, antagonize priming of cytokine secretion by macrophages and dendritic cells. In another embodiment, apoptotic cell supernatants increase Tregs that suppress inflammatory responses and / or prevent excessive release of cytokines.

[0234] In some embodiments, apoptotic cell supernatants, such as apoptotic cell-phagocytic cell supernatants, are administered to inhibit one or more proinflammatory cytokines. In some embodiments, proinflammatory cytokines include IL-1β, IL-6, TNF-α or IFN-γ or any combination thereof. In another embodiment, apoptotic cell supernatants are administered to promote the secretion of one or more anti-inflammatory cytokines. In some embodiments, anti-inflammatory cytokines include TGF-β, IL10 or PGE2 or any combination thereof.

[0235] In another embodiment, administration of apoptotic cell supernatant produces potential tolerogenic dendritic cells, in some embodiments, the cells are capable of migration, and in some embodiments, the migration is due to CCR7. In another embodiment, administration of apoptotic cell supernatant triggers various signaling events, in one embodiment, the signaling event is TAM receptor signaling (Tyro3, ​​Axl and Mer), which in some embodiments inhibits inflammation in antigen presenting cells. In some embodiments, Tyro-3, Axl and Mer constitute the TAM family of receptor tyrosine kinases (RTKs), characterized by conserved sequences within the kinase domain and adhesion molecule-like extracellular domain. In another embodiment, administration of apoptotic cell supernatant activates signaling through MerTK. In another embodiment, administration of apoptotic cell supernatant activates the phosphatidylinositol 3-kinase (PI3K) / AKT pathway, in some embodiments, the pathway negatively regulates NF-κB. In another embodiment, administration of apoptotic cell supernatant negatively regulates inflammasomes, in one embodiment, the inflammasomes lead to inhibition of proinflammatory cytokine secretion, DC maturation or a combination thereof. In another embodiment, administration of apoptotic cell supernatants upregulates the expression of anti-inflammatory genes such as Nr4a, Thbs1, or a combination thereof. In another embodiment, administration of apoptotic cell supernatants induces high levels of AMP, and in some embodiments, AMP accumulates in a pan-connexin 1-dependent manner. In another embodiment, administration of apoptotic cell supernatants inhibits inflammation.

[0236] In some embodiments, a single dose of apoptotic cell supernatant is administered. In some embodiments, multiple doses of apoptotic cell supernatant are administered. In some embodiments, 2 doses of apoptotic cell supernatant are administered. In some embodiments, 3 doses of apoptotic cell supernatant are administered. In some embodiments, 4 doses of apoptotic cell supernatant are administered. In some embodiments, 5 doses of apoptotic cell supernatant are administered. In some embodiments, 6 doses of apoptotic cell supernatant are administered. In some embodiments, 7 doses of apoptotic cell supernatant are administered. In some embodiments, 8 doses of apoptotic cell supernatant are administered. In some embodiments, 9 doses of apoptotic cell supernatant are administered. In some embodiments, more than 9 doses of apoptotic cell supernatant are administered. In some embodiments, multiple doses of apoptotic cell supernatant are administered. In some embodiments, multiple doses of apoptotic cell supernatant are administered during treatment of a subject in need thereof.

[0237] In some embodiments, each dose described herein can be the dose of apoptotic cell supernatant ± about 5%, 10%, 15%, 20% or 25%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 5%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 10%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 15%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 20%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 25%. In some embodiments, the dose of apoptotic cell supernatant is the dose of apoptotic cell supernatant ± about 5%-25%. For example, but not limited to about 100×10 6 -210×10 6 ±20% of the dose, or about 100 × 10 6 ± about 20% of the dose, or 120 × 10 6 ± about 20% of the dose, or 140 × 10 6 ± about 20% of the dose, or 160 × 10 6 ± about 20% of the dose, or 180 × 10 6 ± about 20% of the dose, or 200 × 10 6 ± about 20% of the dose, etc. Similar embodiments include ± 5%, ± 10%, ± 15%, ± 20% or ± 25% of the dose.

[0238] In some embodiments, the dosage of apoptotic cell supernatant is applied every day. In some embodiments, the dosage of apoptotic cell supernatant is applied weekly. In some embodiments, the dosage of apoptotic cell supernatant is applied every half week (twice a week). In some embodiments, the dosage of apoptotic cell supernatant is applied every two weeks (bi-weekly) (every two weeks). In some embodiments, the dosage of apoptotic cell supernatant is applied monthly. In some embodiments, the dosage of apoptotic cell supernatant is applied with an irregular regimen, such as daily application within a given time period, followed by half a week, or weekly, or two weeks, or monthly application, or a combination thereof.

[0239] In some embodiments, apoptotic cell supernatants can be administered by any method known in the art for applying cell supernatants directly to the desired area, including but not limited to injection and infusion.

[0240] In some embodiments, administration comprises injection and / or infusion directly into a joint. In some embodiments, administration comprises injection and / or infusion adjacent to a joint. In some embodiments, administration comprises injection and / or infusion directly at the site of vanishing bone disease. In some embodiments, administration comprises injection and / or infusion near the site of vanishing bone disease. The apoptotic cell supernatant for injection can be in the form of a pharmaceutical composition formulated as a sterile injectable solution.

[0241] In some embodiments, the apoptotic cell supernatant can be administered by topical application, wherein the cells or the pharmaceutical composition comprising the apoptotic cell supernatant are applied directly to the area in need. In some embodiments, administration comprises direct topical application at the joint site. In some embodiments, administration comprises topical application adjacent to the joint. In some embodiments, administration comprises direct topical application at the site of vanishing bone disease. In some embodiments, administration comprises topical application adjacent to the site of vanishing bone disease. The apoptotic cell supernatant for topical application can be in the form of a pharmaceutical composition formulated as a topical ointment, cream, oil, patch, or skin patch. In some embodiments, the apoptotic cell supernatant can be administered by infiltrating cartilage in the area in need, wherein the cells or the pharmaceutical composition comprising the apoptotic cell supernatant are directly targeted to the area in need. In some embodiments, administration comprises infiltrating a joint with the apoptotic cell supernatant or its composition. In some embodiments, administration comprises infiltrating tissue adjacent to the joint with the apoptotic cell supernatant or its composition. In some embodiments, administration comprises infiltrating the area of ​​the vanishing bone disease site with the apoptotic cell supernatant or its composition. In some embodiments, administering comprises infiltrating tissue adjacent to the site of vanishing bone disease with the apoptotic cell supernatant or a composition thereof.

[0242] In some embodiments, application of the apoptotic cell supernatant is topical.

[0243] Composition

[0244] As used herein, the terms "composition" and "pharmaceutical composition" are used interchangeably in some embodiments and have all the same properties and meanings. In some embodiments, disclosed herein are pharmaceutical compositions for treating conditions or diseases described herein. In some embodiments, disclosed herein are pharmaceutical compositions for treating osteoarthritis. In some embodiments, disclosed herein are pharmaceutical compositions for treating vanishing bone disease.

[0245] In some embodiments, the pharmaceutical composition comprises an early apoptotic cell population as described in detail above. In other embodiments, the pharmaceutical composition comprises a supernatant from an early apoptotic cell population as described in detail above. In some embodiments, the pharmaceutical composition comprises an apoptotic supernatant as described in detail above. In some embodiments, the pharmaceutical composition comprises an apoptotic-phagocytic supernatant as described in detail above.

[0246] In yet another embodiment, as described herein, a pharmaceutical composition for treating osteoarthritis and vanishing bone disease comprises an effective amount of an early apoptotic cell population and a pharmaceutically acceptable excipient. In some embodiments, a composition comprising apoptotic cells is used in a method disclosed herein, for example, for treating pain caused by osteoarthritis. In some embodiments, a composition comprising apoptotic cells is used in a method for alleviating pain in osteoarthritis. In some embodiments, a composition comprising apoptotic cells is used in a method for reducing joint inflammation caused by osteoarthritis. In some embodiments, a composition comprising apoptotic cells is used in a method for reducing swelling in or around a joint caused by osteoarthritis. In some embodiments, a composition comprising apoptotic cells is used in a method for inhibiting or slowing progressive degeneration of articular cartilage in a joint. In some embodiments, a composition comprising apoptotic cells is used in a method for inhibiting or slowing progressive erosion of bone tissue caused by vanishing bone disease. In some embodiments, a composition comprising apoptotic cells is used in a method for increasing the movement of a joint affected by osteoarthritis. In some embodiments, a composition comprising apoptotic cells is used in a method for increasing the range of motion of a joint affected by osteoarthritis.

[0247] The dosage of early apoptotic cells is described in detail above. The effective amount of the early apoptotic cell population includes the above dosage, for example but not limited to, the dosage of early apoptotic cells is about 10×10 6 cells ± 20% to 1 × 10 9 In some embodiments, the composition comprises an effective amount of an early apoptotic cell population and a pharmaceutically acceptable excipient.

[0248] In some embodiments, the apoptotic cells contained in the composition include apoptotic cells in an early apoptotic state. In some embodiments, the apoptotic cells contained in the composition are pooled third-party donor cells. In some embodiments, the apoptotic cells contained in the composition are allogeneic donor cells. In some embodiments, the apoptotic cells contained in the composition are autologous donor cells. In some embodiments, the apoptotic cells contained in the composition are pooled autologous donor cells. In some embodiments, the apoptotic cells contained in the composition are irradiated.

[0249] In yet another embodiment, as described herein, a pharmaceutical composition for treating osteoarthritis and vanishing bone disease comprises an effective amount of early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant, and a pharmaceutically acceptable excipient. In some embodiments, a composition comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant is used in a method disclosed herein, for example, for treating pain caused by osteoarthritis. In some embodiments, a composition comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant is used in a method for alleviating osteoarthritis pain. In some embodiments, a composition comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant is used in a method for reducing joint inflammation caused by osteoarthritis. In some embodiments, a composition comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant is used in a method for reducing swelling in or around a joint caused by osteoarthritis. In some embodiments, a composition comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant is used in a method for inhibiting or slowing the progressive degeneration of articular cartilage in a joint. In some embodiments, a composition comprising an early apoptotic cell supernatant or an apoptotic supernatant or an apoptotic-phagocytic cell supernatant is used in a method of inhibiting or slowing the progressive erosion of bone tissue caused by vanishing bone disease. In some embodiments, a composition comprising an early apoptotic cell supernatant or an apoptotic supernatant or an apoptotic-phagocytic cell supernatant is used in a method of increasing the motion of a joint affected by osteoarthritis. In some embodiments, a composition comprising an early apoptotic cell supernatant or an apoptotic supernatant or an apoptotic-phagocytic cell supernatant is used in a method of increasing the range of motion of a joint affected by osteoarthritis.

[0250] The dosage of the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic cell supernatant is described in detail above. The effective amount of the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic cell supernatant includes the above dosages, such as but not limited to the dosage collected from the apoptotic cell culture or apoptotic-phagocytic cell culture, wherein the apoptotic cells are about 1x10 6 cells ± 20% to 1 × 10 9 In some embodiments, the composition comprises an effective amount of early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic cell supernatant and a pharmaceutically acceptable excipient.

[0251] In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains culture medium collected from apoptotic cells in an early apoptotic state. In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains supernatant collected from pooled third-party donor cells. In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains supernatant collected from allogeneic donor cells. In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains supernatant collected from autologous donor cells. In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains supernatant collected from pooled autologous donor cells. In some embodiments, the early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant contained in the composition contains supernatant collected from irradiated cells.

[0252] Those skilled in the art will appreciate that a "pharmaceutical composition" may encompass a preparation of one or more active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0253] Those skilled in the art will understand that the phrases "physiologically acceptable carrier", "pharmaceutically acceptable carrier", "physiologically acceptable excipient" and "pharmaceutically acceptable excipient" can be used interchangeably and can cover carriers, excipients or diluents that do not cause significant irritation to an organism and do not destroy the biological activity and properties of the administered active ingredient.

[0254] The skilled artisan will appreciate that "excipients" can encompass inert substances added to pharmaceutical compositions to further facilitate the administration of the active ingredients. In some embodiments, excipients include calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0255] Techniques for formulation and administration of drugs are found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0256] In some embodiments, the compositions disclosed herein include therapeutic compositions. In some embodiments, the compositions disclosed herein include therapeutic efficacy.

[0257] In some embodiments, the compositions disclosed herein are administered once. In another embodiment, the compositions are administered twice. In another embodiment, the compositions are administered three times. In another embodiment, the compositions are administered four times. In another embodiment, the compositions are administered at least four times. In another embodiment, the compositions are administered more than four times. In another embodiment, the compositions are administered more than once.

[0258] In some embodiments, the compositions disclosed herein are applied daily, weekly, monthly, or at intervals suitable for a particular person's potential disease. In some embodiments, the compositions disclosed herein are applied daily. In some embodiments, the compositions disclosed herein are applied every half week. In some embodiments, the compositions disclosed herein are applied weekly. In some embodiments, the compositions disclosed herein are applied every two weeks. In some embodiments, the compositions disclosed herein are applied monthly. In some embodiments, the compositions disclosed herein are applied using different schemes, such as, but not limited to, first applied daily, and then applied every half week, or weekly, or fortnightly after a period of time. In some embodiments, the compositions disclosed herein are applied at intervals suitable for a particular person's potential disease, such as, but not limited to, osteoarthritis.

[0259] In some embodiments, the composition is injected into a desired site, such as a joint. In some embodiments, the composition is injected near a desired site, such as near a joint. In some embodiments, the composition is injected into a desired site, such as a site of articular cartilage degeneration. In some embodiments, the composition is injected near a desired site, such as a site adjacent to articular cartilage degeneration. In some embodiments, the composition is injected into a desired site, such as a site of bone erosion. In some embodiments, the composition is injected near a desired site, such as near a site of bone erosion.

[0260] In some embodiments, the composition is infused into a desired site, such as a joint. In some embodiments, the composition is infused near a desired site, such as near a joint. In some embodiments, the composition is infused into a desired site, such as a site of articular cartilage degeneration. In some embodiments, the composition is infused near a desired site, such as a site adjacent to articular cartilage degeneration. In some embodiments, the composition is infused into a desired site, such as a site of bone erosion. In some embodiments, the composition is infused near a desired site, such as a site of bone erosion.

[0261] In some embodiments, the composition is infiltrated into a desired site, such as a joint. In some embodiments, articular cartilage is infiltrated by apoptotic cells or a composition thereof at a desired site. In some embodiments, the composition is infiltrated near a desired site, such as a joint adjacent. In some embodiments, the composition is infiltrated into a desired site, such as a site of articular cartilage degeneration. In some embodiments, the composition is infiltrated near a desired site, such as a site of adjacent articular cartilage degeneration. In some embodiments, the composition is infiltrated into a desired site, such as a site of bone erosion. In some embodiments, the composition is infiltrated near a desired site, such as a site of bone erosion.

[0262] In some embodiments, administration of the compositions described herein reduces the release of pro-inflammatory and anti-inflammatory cytokines or chemokines from the synovium, or cartilage, or any joint component, or a combination thereof. In some embodiments, administration of the compositions described herein reduces the release of pro-inflammatory cytokines or chemokines from the synovium, or cartilage, or any joint component, or a combination thereof. In some embodiments, administration of the compositions described herein reduces the release of anti-inflammatory cytokines or chemokines from the synovium, or cartilage, or any joint component, or a combination thereof. In some embodiments, administration of the compositions described herein rebalances the immune response in the joints. In some embodiments, administration of the compositions described herein rebalances the immune response in the synovial fluid present in the joints. In some embodiments, administration of the compositions described herein rebalances the immune response in the joint fluid. In some embodiments, administration of the compositions described herein rebalances the immune response in the intra-articular cartilage. In some embodiments, administration of the compositions described herein rebalances the immune response at the site of vanishing bone disease.

[0263] preparation

[0264] The pharmaceutical composition comprising an early apoptotic cell colony disclosed herein or comprising an early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic supernatant can be conveniently provided as a sterile liquid product, such as an isotonic aqueous solution, a suspension, an emulsion, a dispersion or a viscous composition, which can be buffered to a selected pH. Liquid products are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more convenient to use to a certain extent, especially for injection. On the other hand, viscous compositions can be prepared within a suitable viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions can include a carrier, and the carrier can be a solvent or a dispersion medium containing, for example, water, saline, phosphate buffered saline, a polyol (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.) and a suitable mixture thereof.

[0265] In some embodiments, the composition comprising early apoptotic cells or comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic cell supernatant is contained in lactated Ringer's solution. In some embodiments, the composition comprising early apoptotic cells or comprising early apoptotic cell supernatant or apoptotic supernatant or apoptotic-phagocytic cell supernatant comprises a known buffer solution, such as but not limited to physiological saline or PBS.

[0266] Sterile injectable solution can be prepared by mixing the early apoptotic cell colony described herein or the early apoptotic cell supernatant described herein or apoptotic supernatant or apoptotic-phagocytic supernatant into the required amount of suitable solvent and the other components of different amounts as required, and used to implement the method disclosed herein. Such composition can be mixed with suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose, etc. According to the route of administration and the required product, the composition can contain auxiliary substances, such as wetting agents, dispersants or emulsifiers (such as methylcellulose), pH buffers, gelling agents or viscosity enhancers, preservatives, flavoring agents, coloring agents, etc. Can refer to standard textbooks, such as " REMINGTON'S PHARMACEUTICAL SCIENCE ", 17th edition, 1985, incorporated herein by reference, to prepare suitable products without excessive experiments.

[0267] Various additives that enhance the stability and sterility of the composition can be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Protection against the action of microorganisms can be ensured by a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, etc. The extended absorption of injectable drug forms can be produced by using agents that delay absorption (such as aluminum monostearate and gelatin). However, according to the disclosure herein, any vehicle, diluent or additive used must be compatible with the genetically modified immune response cells or their ancestors.

[0268] The compositions or formulations described herein can be isotonic, i.e., they can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions disclosed herein can be achieved using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes. Sodium chloride can be preferred, particularly for buffers containing sodium ions.

[0269] If necessary, a pharmaceutically acceptable thickening agent may be used to maintain the viscosity of the composition at a selected level.Methylcellulose may be preferred because it is readily and economically available, and is easy to use.

[0270] Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of thickener will depend on selected agent. An important point is to use an amount that can reach selected viscosity. Obviously, the selection of suitable carriers and other additives will depend on the property of definite route of administration and specific dosage form, for example liquid dosage form (for example, whether the composition is formulated into solution, suspension, gel or other liquid forms, for example delayed release form or liquid-filled form).

[0271] Those skilled in the art will recognize that the components of the composition or formulation should be selected to be chemically inert and not affect the viability or efficacy of the early apoptotic cell population described herein for use in the methods disclosed herein. From the present disclosure and the literature cited herein, this is no problem for those skilled in the art of chemical and pharmaceutical principles, or the problem can be easily avoided by reference to standard textbooks or simple experiments (not involving excessive experiments).

[0272] One consideration regarding the therapeutic use of early apoptotic cells disclosed herein is the number of cells necessary to achieve optimal effect. Similarly, a consideration regarding the therapeutic use of supernatants disclosed herein is the number of cells from which the supernatant is collected necessary to achieve optimal effect. In some embodiments, the number of cells to be administered or the number of cells from which the supernatant is collected may vary depending on the subject being treated. In addition, for local administration to a joint, in some embodiments, the size of the joint may be considered.

[0273] In some embodiments, a human subject is administered 10 5 to 10 10 Between 10 6 to 10 9 Between 10 6 and 10 8 In some embodiments, administering to a human subject from 10 5 to 10 10 Between 10 6 to 10 9 Between 10 6 and 10 8 In some embodiments, the number of apoptotic cells used for administration or for collecting the supernatant for administration may include a range.

[0274] In some embodiments, at 1×10 6 -1×10 9 ±20%. In some embodiments, 1×10 6 -1×10 8±20%. In some embodiments, 1×10 6 -1×10 7 ±20%. In some embodiments, 1×10 7 -1×10 9 ±20%. In some embodiments, 1×10 8 -1×10 9 ±20%. In some embodiments, 10×10 6 -500×10 6 ±20%. In some embodiments, 10×10 6 -210×10 6 ±20%, or about 10×10 6 ± 20%, or 12 × 10 6 ± 20% of the dose or collected from 12 × 10 6 ± approximately 20% of the cell dose, or 14 × 10 6 ± 20% of the dose, or collected from 12 × 10 6 ± 20% of the cell dose, or 16 × 10 6 ± 20% of the dose, or collected from 16 × 10 6 ± approximately 20% of the cell dose, or 18 × 10 6 ± 20% of the dose, or collected from 18 × 10 6 ± 20% of the cell dose, or 20 × 10 6 ± 20% of the dose or collected from 20 × 10 6 ± about 20% of the dose of cells. In some embodiments, at 100×10 6 -210×10 6 ±20%, or about 100×10 6 ± 20%, or 120 × 10 6 ± 20% of the dose or collected from 120 × 10 6 ± 20% of the cell dose, or 140 × 10 6 ± 20% of the dose, or collected from 140 × 10 6 ± approximately 20% of the cell dose, or 160 × 10 6 ± 20% of the dose, or collected from 160 × 10 6 ± 20% of the cell dose, or 180 × 10 6 ± 20% of the dose, or collected from 180x10 6 ± 20% of the cell dose, or 200 × 10 6± 20% of the dose or collected from 200 × 10 6 ± 20% of the cell dose, or 1 × 10 6 ± 20% of the dose or collected from 1 × 10 6 ± 20% of the cell dose, or 1 × 10 7 ± 20% of the dose or collected from 1 × 10 7 ± 20% of the cell dose, or 1 × 10 8 ± 20% of the dose or collected from 1 × 10 8 ± 20% of the cell dose, or 1 × 10 9 ± about 20% or collected from 1×10 9 ± approximately 20% of the cell dose.

[0275] In some embodiments, at 1×10 6 -1×10 9 ±5%. In some embodiments, 1×10 6 -1×10 8 ±5%. In some embodiments, 1×10 6 -1×10 7 ±5%. In some embodiments, 1×10 7 -1×10 9 ±5%. In some embodiments, 1×10 8 -1×10 9 ±5%. In some embodiments, 10×10 6 -500×10 6 ±5%. In some embodiments, 10×10 6 -210×10 6 ±5% or about 10×10 6 ±5%, or 12×10 6 ± 5% of the dose or collected from 12 × 10 6 ± 5% of the cell dose, or 14 × 10 6 ± 5% of the dose or collected from 12 × 10 6 ± 5% of the cell dose, or 16 × 10 6 ± 5% of the dose or collected from 16 × 10 6 ± 5% of the cell dose, or 18 × 10 6 ± 5% of the dose or collected from 18 × 10 6 ± 5% of the cell dose, or 20 × 10 6 ± 5% of the dose or collected from 20 × 10 6± about 5% of the dose of cells. In some embodiments, at 100×10 6 -210×10 6 ±5%, or about 100×10 6 ± 5% or 120 × 10 6 ± 5% of the dose or collected from 120 × 10 6 ± 5% of the cell dose, or 140 × 10 6 ± 5% of the dose, or collected from 140 × 10 6 ± 5% of the cell dose, or 160 × 10 6 ± 5% of the dose, or collected from 160 × 10 6 ±5% of the cell dose, or 180 × 10 6 ± 5% of the dose or collected from 180 × 10 6 ± 5% of the cell dose, or 200 × 10 6 ± 5% of the dose or collected from 200 × 10 6 ± 5% of the cell dose, or 1 × 10 6 ± 5% of the dose or collected from 1 × 10 6 ± 5% of the cell dose, or 1 × 10 7 ± 5% of the dose or collected from 1 × 10 7 ± 5% of the cell dose, or 1 × 10 8 ± 5% of the dose, or collected from 1 × 10 8 ± 5% of the cell dose, or 1 × 10 9 ± 5% of the dose, or collected from 1 × 10 9 ± approximately 5% of the cell dose.

[0276] In some embodiments, at 1×10 6 -1×10 9 ±10%. In some embodiments, 1×10 6 -1×10 8 ±10%. In some embodiments, 1×10 6 -1×10 7 ±10%. In some embodiments, 1×10 7 -1×10 9 ±10%. In some embodiments, 1×10 8 -1×10 9 ±10%. In some embodiments, 10×10 6 -500×10 6±10%. In some embodiments, 10×10 6 -210×10 6 ±10% or about 10×10 6 ± about 10%, or 12×10 6 ± 10% of the dose or collected from 12 × 10 6 ± approximately 10% of the cell dose, or 14 × 10 6 ± about 10% of the dose, or collected from 12 × 10 6 ± approximately 10% of the cell dose, or 16 × 10 6 ± about 10% of the dose or collected from 16 × 10 6 ± approximately 10% of the cell dose, or 18 × 10 6 ± about 10% of the dose or collected from 18 × 10 6 ± approximately 10% of the cell dose, or 20 × 10 6 ± about 10% of the dose or collected from 20×10 6 ± about 10% of the dose of cells. In some embodiments, at 100×10 6 -210×10 6 ±10%, or about 100×10 6 ± about 10%, or 120×10 6 ± about 10% of the dose or collected from 120 × 10 6 ± approximately 10% of the cell dose, or 140 × 10 6 ± about 10% of the dose or collected from 140×10 6 ± approximately 10% of the cell dose, or 160 × 10 6 ± about 10% of the dose or collected from 160 × 10 6 ± approximately 10% of the cell dose, or 180 × 10 6 ± about 10% of the dose or collected from 180 × 10 6 ± approximately 10% of the cell dose, or 200 × 10 6 ± about 10% of the dose or collected from 200 × 10 6 ± about 10% of the cell dose, or 1 × 10 6 ± about 10% of the dose or collected from 1×10 6 ± about 10% of the cell dose, or 1 × 10 7 ± about 10% of the dose or collected from 1×10 7 ± about 10% of the cell dose, or 1 × 10 8 ± about 10% of the dose or collected from 1×10 8 ± about 10% of the cell dose, or 1 × 10 9± about 10% of the dose or collected from 1×10 9 ± approximately 10% of the cell dose.

[0277] In some embodiments, at 1×10 6 to 1×10 9 ±15%. In some embodiments, 1×10 6 to 1×10 8 ±15%. In some embodiments, 1×10 6 -1×10 7 ±15%. In some embodiments, 1×10 7 -1×10 9 ±15%. In some embodiments, 1×10 8 -1×10 9 ±15%. In some embodiments, 10×10 6 -500×10 6 ±15%. In some embodiments, 10×10 6 -210×10 6 ±15%, or about 10×10 6 ± about 15%, or 12×10 6 ± about 15% of the dose or collected from 12 × 10 6 ± approximately 15% of the cell dose, or 14 × 10 6 ± about 15% of the dose or collected from 12 × 10 6 ± approximately 15% of the cell dose, or 16 × 10 6 ± about 15% of the dose or collected from 16 × 10 6 ± approximately 15% of the cell dose, or 18 × 10 6 ± about 15% of the dose or collected from 18 × 10 6 ± approximately 15% of the cell dose, or 20 × 10 6 ± about 15% of the dose or collected from 20×10 6 ± about 15% of the dose of cells. In some embodiments, at 100×10 6 -210×10 6 ±15% or about 100×10 6 ± about 15%, or 120×10 6 ± about 15% of the dose or collected from 120 × 10 6 ± approximately 15% of the cell dose, or 140 × 10 6 ± about 15% of the dose or collected from 140×10 6± approximately 15% of the cell dose, or 160 × 10 6 ± about 15% of the dose or collected from 160×10 6 ± approximately 15% of the cell dose, or 180 × 10 6 ± 15% of the dose or collected from 180 × 10 6 ± approximately 15% of the cell dose, or 200 × 10 6 ± 15% of the dose or collected from 200 × 10 6 ± about 15% of the cell dose, or 1 × 10 6 ± about 15% of the dose or collected from 1×10 6 ± about 15% of the cell dose, or 1 × 10 7 ± about 15% of the dose or collected from 1×10 7 ± about 15% of the cell dose, or 1 × 10 8 ± about 15% of the dose or collected from 1x1×10 8 ± about 15% of the cell dose, or 1 × 10 9 ± about 15% of the dose or collected from 1×10 9 The dosage of ± about 15% of the cells. More effective cells or cell supernatants can be administered in even smaller quantities or collected from fewer cells, respectively. The precise determination of what is considered an effective dosage can be based on individual factors for each subject, including their size, age, sex, weight, the condition of the particular subject, and the site of application, such as the size of the synovial joint or bone area being treated. The dosage can be readily determined by one skilled in the art from this disclosure and the knowledge in the art.

[0278] Those skilled in the art can easily determine the quantity of cells or the amount of supernatant in the composition used in the method disclosed herein, and optional additives, vehicles and / or carriers. Typically, any additive (except active cells and / or agents) exists in phosphate buffered saline with the amount of 0.001% to 50% (weight) solution, and active ingredient exists with microgram to milligram magnitude, for example, about 0.0001wt% to about 5wt%. In another embodiment, about 0.0001wt% to about 1wt%. In another embodiment, about 0.0001wt% to about 0.05wt% or about 0.001wt% to about 20wt%. In other embodiments, about 0.01wt% to about 10wt%. In another embodiment, about 0.05wt% to about 5wt%. Of course, for any composition to be administered to an animal or human, and for any particular method of administration, it is preferred that the toxicity, for example, by determining the lethal dose (LD) and LD50 in a suitable animal model, such as a rodent, such as a mouse; and the dose of the composition that elicits an appropriate response, the concentration of the components therein, and the timing of administering the composition. Such determination does not require undue experimentation based on the knowledge of those skilled in the art, the present disclosure, and the literature cited herein. Moreover, the duration of continuous administration can be determined without undue experimentation.

[0279] How to use

[0280] In some embodiments, disclosed herein is a method of treating a joint disease or disorder in a subject in need thereof, the method comprising the step of administering a composition comprising a population of early apoptotic cells directly into a joint of the subject, wherein the administration treats the joint disease or disorder in the subject.

[0281] The skilled artisan will appreciate that the term "joint disease or disorder" encompasses diseases in which the normal function or use of a joint is impaired. Impaired or lost function may result in discomfort or pain in or around a joint. Joint diseases and disorders may affect the bones, joint capsules, cartilage, tendons, ligaments, tendon sheaths, bursae, synovial fluid in or around the affected joint. Common joint diseases include osteoarthritis, rheumatoid arthritis, gout, lupus, tendinitis, bursitis, carpal tunnel syndrome, sprains, and the like. In some embodiments, a joint disease or disorder includes osteoarthritis.

[0282] In some embodiments, the methods for treating osteoarthritis disclosed herein treat inflammatory osteoarthritis. In some embodiments, the methods for treating osteoarthritis disclosed herein treat erosive osteoarthritis (EOA). In some embodiments, the methods for treating osteoarthritis disclosed herein treat Gorham disease with inflammatory osteoarthritis. In some embodiments, osteoarthritis includes inflammatory arthritis, erosive osteoarthritis and vanishing bone disease. In some embodiments, osteoarthritis includes any combination of inflammatory arthritis, erosive osteoarthritis and vanishing bone disease. In some embodiments, osteoarthritis includes inflammatory arthritis, or erosive osteoarthritis, or vanishing bone disease, or a combination thereof.

[0283] In some embodiments, treatment methods comprising directly administering early apoptotic cells also help treat vanishing bone disease. In some embodiments, disclosed herein is a method of treating osteoarthritis or vanishing bone disease, or a combination thereof, in a subject in need thereof, the method comprising the step of administering a composition comprising a population of early apoptotic cells directly into a joint of the subject or at a site of vanishing bone disease, wherein the administration treats osteoarthritis or vanishing bone disease, respectively, in the subject. In some embodiments, disclosed herein is a method of treating osteoarthritis or vanishing bone disease, or a combination thereof, in a subject in need thereof, the method comprising the step of administering a composition comprising an early apoptotic cell supernatant, an apoptotic cell supernatant, or an apoptotic cell-phagocyte supernatant directly into a joint of the subject or at a site of vanishing bone disease, wherein the administration treats osteoarthritis or vanishing bone disease, or a combination thereof, respectively, in the subject.

[0284] In some embodiments, treatment of osteoarthritis or vanishing bone disease, or a combination thereof, comprises reducing pain, reducing inflammation, reducing swelling, inhibiting progressive degeneration of articular cartilage, reducing progressive degeneration of articular cartilage, inhibiting erosion of bone tissue or slowing erosion of bone tissue, reducing bone fractures, reducing broken bones, inhibiting bone fractures, inhibiting broken bones, improving the quality of life of the subject with osteoarthritis or vanishing bone disease, or any combination thereof. In some embodiments, treatment of osteoarthritis or vanishing bone disease, or a combination thereof, using the methods described herein, rebalances the immune response within the joint or bone tissue, or within tissue adjacent thereto, or any combination thereof.

[0285] In some embodiments, the therapeutic methods disclosed herein provide long-term treatment. In some embodiments, the effect of treatment lasts for at least one month after administration. In some embodiments, the effect of treatment lasts for at least two months after administration. In some embodiments, the effect of treatment lasts for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months or 11 months after administration. In some embodiments, the effect of treatment lasts for at least 1-6 months after administration. In some embodiments, the effect of treatment lasts for at least one month and one year after administration. In some embodiments, the effect of treatment lasts for at least one year after administration. In some embodiments, the effect of treatment lasts for more than one year after administration.

[0286] In some embodiments, disclosed herein is a method for treating osteoarthritis in a subject in need, the method comprising administering a composition comprising an early apoptotic cell colony directly to the subject's joint, wherein the administration treats the subject's osteoarthritis. In some embodiments, disclosed herein is a method for treating osteoarthritis in a subject in need, the method comprising administering a composition comprising an early apoptotic cell supernatant, an apoptotic cell supernatant, or an apoptotic cell-phagocytic cell supernatant directly to the subject's joint, wherein the administration treats the subject's osteoarthritis. In some embodiments, osteoarthritis includes degenerative osteoarthritis. In some embodiments, osteoarthritis includes inflammatory osteoarthritis. In some embodiments, osteoarthritis includes erosive osteoarthritis (EOA). In some embodiments, osteoarthritis includes Gorham's vanishing bone disease with inflammatory osteoarthritis.

[0287] Osteoarthritis commonly affects synovial joints, such as those in the knees, hips, shoulders, elbows, ankles, wrists, fingers, thumbs, neck, toes, thumbs, hands, feet, and spine. Synovial joints consist of two bone ends covered by articular cartilage. Osteoarthritis may be caused by injuries to the menisci or ligaments, suppurative infections, ligament instability, joint fractures, obesity, or natural degenerative causes. Osteoarthritis generally involves progressive degeneration of the articular cartilage at or within these joints. Joints affected by osteoarthritis may be painful, inflamed, swollen, and have a reduced range of motion.

[0288] Examples of the effects of osteoarthritis include, but are not limited to, the following: Hip osteoarthritis can cause pain, stiffness, and severe disability. Patients may experience pain in the hip, groin, inner thigh, buttocks, or knees. Finger osteoarthritis may cause the fingers to become enlarged and rough. The disease may cause small bony protrusions to appear at the end joints of the fingers. The affected fingers may be painful or stiff and numb. More women than men suffer from finger osteoarthritis, and they especially develop it after menopause. The base of the thumb joint may also be affected by osteoarthritis. Osteoarthritis of the neck and spine may cause stiffness and pain in the neck or lower back. It may also cause weakness or numbness in the arms or legs. Osteoarthritis of the neck and spine is often debilitating and may cause the patient to be bedridden.

[0289] One of the most common sites for osteoarthritis to develop is the knee. Knee osteoarthritis may cause stiffness, swelling, and pain in the knee—making it difficult to walk, climb, and get in and out of chairs and the bathtub. If left untreated, knee osteoarthritis can lead to permanent disability and reduced quality of life.

[0290] In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a synovial joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a synovial joint selected from a knee joint, a hip joint, a shoulder joint, a joint between the cervical vertebrae, an elbow joint, an ankle joint, a wrist joint, a finger joint, a toe joint, a hand joint, a foot joint, a thumb joint, or a combination thereof. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a knee joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a hip joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a shoulder joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a cervical intervertebral joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in an elbow joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in an ankle joint. In some embodiments, the method of treating osteoarthritis in a subject in need includes treating osteoarthritis in a wrist joint. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in the finger joints. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in the toe joints. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in the hand joints. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in the foot joints. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in the thumb joints. In some embodiments, methods of treating osteoarthritis in a subject in need thereof include treating osteoarthritis in a combination of synovial joints.

[0291] Those skilled in the art will appreciate that the term "treatment" and its grammatical variants may encompass both therapeutic treatments and prophylactic or preventative measures, wherein the purpose is to prevent or alleviate the target pathological condition or disorder of osteoarthritis or vanishing bone disease, or a combination thereof. In some embodiments, treatment may include directly affecting or curing, suppressing, inhibiting, preventing, reducing severity, delaying onset, alleviating symptoms associated with osteoarthritis or vanishing bone disease, or a combination thereof. In some embodiments, "treatment" may encompass delaying progression, accelerating recovery, improving efficacy, or reducing resistance to alternative therapies, or a combination thereof. In some embodiments, "prevention" includes delaying the onset of symptoms, preventing recurrence of osteoarthritis or vanishing bone disease, or a combination thereof, reducing the number or frequency of recurring episodes, increasing the latency between symptom onsets, or a combination thereof. In some embodiments, "suppression" or "inhibition" includes reducing the severity of symptoms, reducing the severity of acute episodes, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

[0292] In some embodiments, treating osteoarthritis includes reducing pain, reducing inflammation, reducing swelling, inhibiting progressive degeneration of articular cartilage, reducing progressive degeneration of articular cartilage, or any combination thereof in and / or around the treated joint. Joint function can be measured by assessing parameters such as the presence or absence of discomfort or pain during range of motion and movement, or by assessing the range of motion itself.

[0293] In some embodiments, treating osteoarthritis comprises reducing pain in and / or around the treated joint. In some embodiments, treatment reduces local pain around the joint. In some embodiments, relieving or reducing pain can be associated with a reduction or relief of pain during joint movement. In some embodiments, relieving or reducing pain can be associated with a reduction or relief of joint pain under static conditions. In some embodiments, treating osteoarthritis comprises reducing stiffness in and / or around the treated joint.

[0294] In some embodiments, the reduction or relief of pain allows for increased range of motion of a joint. In some embodiments, osteoarthritis is treated using the methods disclosed herein for direct administration of early apoptotic cells or apoptotic supernatants, increasing the motion of the treated joint. In some embodiments, the increased motion comprises an increased range of motion or the increased motion is accompanied by reduced pain, or a combination thereof. In some embodiments, the increased motion comprises an increased range of motion. In some embodiments, the increased motion is accompanied by reduced pain.

[0295] In some embodiments, treating osteoarthritis includes reducing inflammation in and / or around the treated joint. In some embodiments, the treatment that leads to reduced inflammation can be the result of reduced secretion of proinflammatory cytokines and chemokines. For example, but not limited to the reduction of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα or a combination thereof. In some embodiments, treating osteoarthritis by directly administering early apoptotic cells reduces proinflammatory and anti-inflammatory cytokines and chemokines in the synovial fluid associated with the treated joint. In some embodiments, the proinflammatory cytokines and chemokines reduced after treatment with early apoptotic cells or apoptotic supernatants include any proinflammatory cytokines or chemokines.

[0296] The skilled person will recognize that the therapeutic effect can be measured by measuring the level of cytokines and / or chemokines in the synovial fluid associated with the treated joint. Methods for measuring the presence and concentration of cytokines and / or chemokines are well known in the art, and any known method can be used. In some embodiments, synovial fluid will be collected to measure different cytokines and / or chemokines in the joint fluid. In some embodiments, synovial fluid will be collected to measure different metalloproteinases in the joint fluid. In some embodiments, synovial fluid will be collected to measure different proinflammatory cytokines in the joint fluid. In some embodiments, synovial fluid will be collected to measure different anti-inflammatory cytokines in the joint fluid. In some embodiments, synovial fluid will be collected to measure different proinflammatory and anti-inflammatory cytokines in the joint fluid. In some embodiments, synovial fluid will be collected to measure any one of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα in the joint fluid or any combination thereof.

[0297] In some embodiments, synovial fluid is collected and analyzed prior to treatment. In some embodiments, synovial fluid is collected and analyzed prior to and during treatment. In some embodiments, synovial fluid is collected and analyzed prior to treatment, during treatment, and as a follow-up after treatment is completed.

[0298] In some embodiments, a synovial biopsy will be performed prior to treatment. In some embodiments, a synovial biopsy will be performed prior to and during treatment. In some embodiments, a synovial biopsy will be performed prior to and during treatment, and as a follow-up after treatment is completed.

[0299] In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or apoptotic supernatant reduces the level of pro-inflammatory and anti-inflammatory cytokines or chemokines in the synovial fluid associated with the treated joint. In some embodiments, after treatment with early apoptotic cells or with apoptotic cell supernatant, pro-inflammatory cytokines and chemokines are reduced, and the pro-inflammatory cytokines and chemokines include any one of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα, or any combination thereof. In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of pro-inflammatory and anti-inflammatory cytokines or chemokines in the synovial fluid associated with the treated joint. In some embodiments, the proinflammatory cytokines and chemokines whose concentrations are altered following treatment with early apoptotic cells include any of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9, or TNFα, or any combination thereof.

[0300] In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-1 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-6 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-8 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-10 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-1β in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-2 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-15 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-22 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IL-9 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of MIP-1β in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of MCP-1 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of MDC in the synovial fluid associated with the treated joint. In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of IP-10 in the synovial fluid associated with the treated joint. In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of fractalkine in the synovial fluid associated with the treated joint.In some embodiments, methods of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant reduces the level of TNFα in the synovial fluid associated with the treated joint.

[0301] In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-1 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-6 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-8 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-10 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-1β in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-2 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-15 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IL-22 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of MIP-1β in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of MCP-1 in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of MDC in the synovial fluid associated with the treated joint. In some embodiments, the method for treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of IP-10 in the synovial fluid associated with the treated joint. In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of fractalkine in the synovial fluid associated with the treated joint. In some embodiments, the method of treating osteoarthritis by directly administering a composition comprising early apoptotic cells or comprising apoptotic cell supernatant changes the level of TNFα in the synovial fluid associated with the treated joint.

[0302] In some embodiments, the cytokine or chemokine is reduced by about 10%-90%. In some embodiments, the cytokine or chemokine is reduced by about 20%-80%. In some embodiments, the cytokine or chemokine is reduced by about 30%-70%. In some embodiments, the cytokine or chemokine is reduced by about 40%-60%. In some embodiments, the cytokine or chemokine is reduced by about 80%-99%. In some embodiments, the cytokine or chemokine is reduced by about 10%. In some embodiments, the cytokine or chemokine is reduced by about 20%. In some embodiments, the cytokine or chemokine is reduced by about 30%. In some embodiments, the cytokine or chemokine is reduced by about 40%. In some embodiments, the cytokine or chemokine is reduced by about 50%. In some embodiments, the cytokine or chemokine is reduced by about 60%. In some embodiments, the cytokine or chemokine is reduced by about 70%. In some embodiments, the cytokine or chemokine is reduced by about 80%. In some embodiments, the cytokine or chemokine is reduced by about 90%. In some embodiments, the cytokine or chemokine is reduced by about 95%. In some embodiments, the cytokine or chemokine is reduced by about 99%.

[0303] Those skilled in the art will appreciate that each cytokine or chemokine can be reduced by different percentages, wherein the reduction of each cytokine or chemokine is about 10%-90%. In some embodiments, the cytokine or chemokine is reduced by about 20%-80%. In some embodiments, each cytokine or chemokine is reduced by about 30%-70%. In some embodiments, each cytokine or chemokine is reduced by about 40%-60%. In some embodiments, each cytokine or chemokine is reduced by about 80%-99%. In some embodiments, each cytokine or chemokine is reduced by about 10%. In some embodiments, each cytokine or chemokine is reduced by about 20%. In some embodiments, each cytokine or chemokine is reduced by about 30%. In some embodiments, each cytokine or chemokine is reduced by about 40%. In some embodiments, each cytokine or chemokine is reduced by about 50%. In some embodiments, each cytokine or chemokine is reduced by about 60%. In some embodiments, each cytokine or chemokine is reduced by about 70%. In some embodiments, each cytokine or chemokine is reduced by about 80%. In some embodiments, each cytokine or chemokine is reduced by about 90%. In some embodiments, each cytokine or chemokine is reduced by about 95%. In some embodiments, each cytokine or chemokine is reduced by about 99%.

[0304] In some embodiments, treating osteoarthritis comprises reducing swelling in and / or around the treated joint. In some embodiments, treating osteoarthritis comprises inhibiting progressive degeneration of articular cartilage in the treated joint. In some embodiments, treating osteoarthritis comprises reducing progressive degeneration of articular cartilage in the treated joint.

[0305] Those skilled in the art will appreciate that the size of the cartilage can be measured using ultrasound or MRI or X-ray analysis or a combination thereof to measure the inhibition of progressive degeneration. In addition, pain scales for the degree of pain felt by a subject in the art can be used to assess the benefits of treatment and the alleviation of pain. In addition, fluid analysis can be performed by analyzing the different components of a test sample including, for example, but not limited to, synovial fluid or joint fluid, and measuring cytokine levels, or chemokine levels, or metalloproteinase levels, or any combination thereof.

[0306] In some embodiments, the method for treating osteoarthritis in a subject in need includes a step of directly administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant to a joint of the subject, wherein the administration treats osteoarthritis. In some embodiments, the joint comprises a synovial joint. In some embodiments, the synovial joint comprises a knee joint, a hip joint, a shoulder joint, a joint between the cervical vertebrae, an elbow joint, an ankle joint, a wrist joint, a finger joint, a toe joint or a thumb joint, a hand joint, a foot joint, or a combination thereof. In some embodiments, a subject in need suffers from the pain of a single joint. In some embodiments, a subject in need suffers from the pain of more than one joint.

[0307] In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a synovial joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a knee joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a hip joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a shoulder joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a joint between cervical vertebrae. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into an elbow joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into an ankle joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a wrist joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises directly administering it into a finger joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises administering directly into a toe joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises administering directly into a thumb joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises administering directly into a hand joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises administering directly into a foot joint. In some embodiments, administering a composition comprising an early apoptotic cell colony or comprising an apoptotic cell supernatant comprises administering directly into a synovial joint selected from a knee joint, a hip joint, a shoulder joint, a cervical intervertebral joint, an elbow joint, an ankle joint, a wrist joint, a finger joint, a toe joint, a hand joint, a foot joint, or a thumb joint, or a combination thereof.

[0308] Those skilled in the art will understand that the term "directly" encompasses administration of early apoptotic cells or apoptotic cell supernatant, or a composition thereof, within or immediately adjacent to a treated joint, respectively. Direct treatment at the joint is distinct from systemic administration, such as, but not limited to, intravenous injection or intraperitoneal infusion. In some embodiments, administration of a composition comprising an early apoptotic cell population or comprising apoptotic cell supernatant for treating osteoarthritis does not include systemic administration.

[0309] In some embodiments, in the methods disclosed herein, direct administration comprises injecting a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into a joint. In some embodiments, in the methods disclosed herein, direct administration comprises injecting a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into a tissue adjacent to a joint. In some embodiments, in the methods disclosed herein, direct administration comprises infusing a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into a joint. In some embodiments, in the methods disclosed herein, direct administration comprises infusing a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into a tissue adjacent to a joint.

[0310] In some embodiments, in the methods disclosed herein, direct administration comprises infiltrating a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into cartilage tissue at a joint. In some embodiments, in the methods disclosed herein, direct administration comprises infiltrating a composition of early apoptotic cells or a composition comprising apoptotic cell supernatant into cartilage tissue adjacent to a joint.

[0311] Vanishing bone disease, also known as Gorham-Stout syndrome, vanishing bone disease, or massive osteolysis, is a rare disease of unknown etiology characterized by progressive bone loss with the destruction of bone matrix and hyperplasia of vascular structures. Subjects with vanishing bone disease experience erosion of bone tissue over time.

[0312] In some embodiments, the methods of treatment disclosed herein include alleviating pain, reducing inflammation, reducing swelling, inhibiting progressive degeneration of articular cartilage, reducing progressive degeneration of articular cartilage, inhibiting erosion of bone tissue or slowing down erosion of bone tissue, or any combination thereof. Affected individuals experience gradual destruction and resorption of bones. Multiple bones may be involved. Commonly affected areas include ribs, spine, pelvis, skull, collarbone (clavicle), shoulder and jaw. Pain and swelling may occur in the affected area. Affected bones are prone to bone loss (osteopenia) and fractures. The severity may vary from person to person, and the disease may cause deformity and functional disability in the affected area.

[0313] In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating vanishing bone disease present in any one or combination of the shoulder, skull, pelvic girdle or a portion thereof, jaw, one or more ribs, clavicle or a portion thereof, or spine. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the shoulder. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the skull. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the pelvic girdle or a portion thereof. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the jaw. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating one or more ribs. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the clavicle or a portion thereof. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating the spine. In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises treating a combination of bones including the shoulder, the skull, the pelvic girdle or a portion thereof, the jaw, one or more ribs, the clavicle or a portion thereof, or the spine.

[0314] In some embodiments, treating vanishing bone disease includes reducing pain, reducing inflammation, reducing swelling, inhibiting erosion of bone tissue, reducing erosion of bone tissue, inhibiting fractures at the disease site, reducing fractures at the disease site, inhibiting bone damage at the disease site, reducing bone damage at the disease site, inhibiting bone resorption at the disease site, reducing bone resorption at the disease site, inhibiting bone loss at the disease site, slowing bone loss at the disease site, inhibiting loss of bone density at the disease site, reducing loss of bone density at the disease site, or any combination thereof. Measurements related to bone mass and bone density can include any method known in the art.

[0315] In some embodiments, treating vanishing bone disease comprises alleviating pain in and / or around the treated bone. In some embodiments, treatment relieves localized pain around the bone. In some embodiments, alleviating or reducing pain can be associated with a reduction or relief of pain during bone movement or in a bone region. In some embodiments, alleviating or reducing pain can be associated with a reduction or relief of pain in a bone under static conditions. In some embodiments, treating osteoarthritis comprises reducing stiffness in or around the treated bone.

[0316] In some embodiments, the reduction or relief of pain allows for increased range of motion of a bone or an area adjacent to a bone. In some embodiments, using the methods disclosed herein to directly administer early apoptotic cells to treat vanishing bone disease increases the motion of the treated bone or an area adjacent to the treated bone. In some embodiments, the increased motion includes an increased range of motion or the increased motion is accompanied by reduced pain, or a combination thereof. In some embodiments, the increased motion includes an increased range of motion. In some embodiments, the increase in increased motion is accompanied by reduced pain.

[0317] In some embodiments, treating vanishing bone disease includes reducing inflammation in and / or around the treated bone. In some embodiments, treatment resulting in reduced inflammation can be the result of reduced secretion of anti-inflammatory or pro-inflammatory cytokines and chemokines or a combination thereof. For example, but not limited to reducing the reduction of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα or any combination thereof. In some embodiments, vanishing bone disease is treated by directly administering early apoptotic cells or apoptotic cell supernatants, reducing pro-inflammatory cytokines and chemokines in tissues adjacent to the treated bone. In some embodiments, the pro-inflammatory cytokines and chemokines reduced after treatment with early apoptotic cells or apoptotic cell supernatants include any pro-inflammatory cytokines or chemokines. In some embodiments, the proinflammatory cytokines and chemokines that are reduced following treatment with early apoptotic cells or apoptotic cell supernatants include any one of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9, or TNFα, or any combination thereof.

[0318] In some embodiments, the cytokine or chemokine is reduced by about 10%-90%. In some embodiments, the cytokine or chemokine is reduced by about 20%-80%. In some embodiments, the cytokine or chemokine is reduced by about 30%-70%. In some embodiments, the cytokine or chemokine is reduced by about 40%-60%. In some embodiments, the cytokine or chemokine is reduced by about 80%-99%. In some embodiments, the cytokine or chemokine is reduced by about 10%. In some embodiments, the cytokine or chemokine is reduced by about 20%. In some embodiments, the cytokine or chemokine is reduced by about 30%. In some embodiments, the cytokine or chemokine is reduced by about 40%. In some embodiments, the cytokine or chemokine is reduced by about 50%. In some embodiments, the cytokine or chemokine is reduced by about 60%. In some embodiments, the cytokine or chemokine is reduced by about 70%. In some embodiments, the cytokine or chemokine is reduced by about 80%. In some embodiments, the cytokine or chemokine is reduced by about 90%. In some embodiments, the cytokine or chemokine is reduced by about 95%. In some embodiments, the cytokine or chemokine is reduced by about 99%.

[0319] Those skilled in the art will appreciate that each cytokine or chemokine can be reduced by different percentages, wherein the reduction of each cytokine or chemokine is about 10%-90%. In some embodiments, the cytokine or chemokine is reduced by about 20%-80%. In some embodiments, each cytokine or chemokine is reduced by about 30%-70%. In some embodiments, each cytokine or chemokine is reduced by about 40%-60%. In some embodiments, each cytokine or chemokine is reduced by about 80%-99%. In some embodiments, each cytokine or chemokine is reduced by about 10%. In some embodiments, each cytokine or chemokine is reduced by about 20%. In some embodiments, each cytokine or chemokine is reduced by about 30%. In some embodiments, each cytokine or chemokine is reduced by about 40%. In some embodiments, each cytokine or chemokine is reduced by about 50%. In some embodiments, each cytokine or chemokine is reduced by about 60%. In some embodiments, each cytokine or chemokine is reduced by about 70%. In some embodiments, each cytokine or chemokine is reduced by about 80%. In some embodiments, each cytokine or chemokine is reduced by about 90%. In some embodiments, each cytokine or chemokine is reduced by about 95%. In some embodiments, each cytokine or chemokine is reduced by about 99%.

[0320] In some embodiments, treating vanishing bone disease includes reducing inflammation in and / or around the treated bone. In some embodiments, treatment resulting in reduced inflammation can be the result of regulating the secretion of proinflammatory cytokines and chemokines. For example, but not limited to regulating the secretion of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9 or TNFα or any combination thereof. In some embodiments, vanishing bone disease is treated by directly administering early apoptotic cells or apoptotic cell supernatants, which changes the proinflammatory cytokines and chemokines in tissues adjacent to the treated bone. In some embodiments, the proinflammatory cytokines and chemokines that are changed after treatment with early apoptotic cells or apoptotic cell supernatants include any proinflammatory cytokines or chemokines. In some embodiments, the proinflammatory cytokines and chemokines that are altered following treatment with early apoptotic cells or apoptotic cell supernatants include any of IL-1, IL-6, IL-8, IL-10, IL-1β, IL-2, IL-15, IL-22, MIP-1β, MCP-1, MDC, IP-10, fractalkine, IL-9, or TNFα, or any combination thereof.

[0321] In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one proinflammatory cytokine or chemokine in the synovial fluid present in the joint. In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one proinflammatory cytokine or chemokine secreted from cells adjacent to the affected bone. In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one anti-inflammatory cytokine or chemokine in the synovial fluid present in the joint. In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one anti-inflammatory cytokine or chemokine secreted by cells adjacent to the affected bone. In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one proinflammatory cytokine or chemokine and one anti-inflammatory cytokine or chemokine in the synovial fluid present in the joint. In some embodiments, the method of administering a composition of early apoptotic cells or apoptotic cell supernatants reduces the concentration of at least one proinflammatory cytokine or chemokine and one anti-inflammatory cytokine or chemokine secreted by cells adjacent to the affected bone.

[0322] In some embodiments, treating vanishing bone disease comprises reducing swelling in and / or around the treated bone.

[0323] In some embodiments, treating vanishing bone disease comprises inhibiting progressive erosion of the treated bone. In some embodiments, erosion may result in loss of bone mass, loss of bone density, and increased occurrence of fractures or breaks in the affected bone. In some embodiments, treating vanishing bone disease comprises reducing progressive erosion of the treated bone. In some embodiments, treating vanishing bone disease comprises reducing erosion of bone tissue. In some embodiments, treating vanishing bone disease comprises inhibiting fractures at the disease site. In some embodiments, treating vanishing bone disease comprises reducing fractures at the disease site. In some embodiments, treating vanishing bone disease comprises inhibiting bone breaks at the disease site. In some embodiments, treating vanishing bone disease comprises reducing bone breaks at the disease site. In some embodiments, treating vanishing bone disease comprises inhibiting bone resorption at the disease site. In some embodiments, treating vanishing bone disease comprises reducing bone resorption at the disease site. In some embodiments, treating vanishing bone disease comprises inhibiting loss of bone mass at the disease site. In some embodiments, treating vanishing bone disease comprises slowing loss of bone mass at the disease site. In some embodiments, treating vanishing bone disease comprises inhibiting loss of bone density at the disease site. In some embodiments, treating vanishing bone disease comprises reducing bone density loss at the disease site. In some embodiments, treating vanishing bone disease comprises a combination of reducing pain, reducing inflammation, reducing swelling, inhibiting bone tissue erosion, reducing bone tissue erosion, inhibiting bone fracture at the disease site, reducing bone fracture at the disease site, inhibiting bone fracture at the disease site, reducing bone fracture at the disease site, inhibiting bone resorption at the disease site, reducing bone resorption at the disease site, inhibiting bone loss at the disease site, slowing bone loss at the disease site, inhibiting bone density loss at the disease site, and reducing bone density loss at the disease site.

[0324] In some embodiments, a method of treating vanishing bone disease in a subject in need thereof comprises the step of administering a composition comprising a population of early apoptotic cells or comprising apoptotic cell supernatant directly to an affected bone site in the subject, wherein the administration treats vanishing bone disease. In some embodiments, the bone comprises a shoulder, a skull, a pelvic girdle or a portion thereof, a jaw, one or more ribs, a clavicle or a portion thereof, or a spine, or a combination thereof.

[0325] In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the shoulder bone, the skull, the pelvic girdle or a portion thereof, the jaw, one or more than one rib, the clavicle or a portion thereof, or the spine, or a combination thereof. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the shoulder bone. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the skull. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the pelvic girdle or a portion thereof. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the jaw or a portion thereof. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of one or more than one rib. In some embodiments, the administration of a composition comprising an early apoptotic cell population or comprising an apoptotic cell supernatant comprises direct administration at the site of the clavicle or a portion thereof. In some embodiments, administering a composition comprising an early apoptotic cell population or comprising apoptotic cell supernatant comprises direct administration at a site of the spine or a portion thereof. In some embodiments, administering a composition comprising an early apoptotic cell population or comprising apoptotic cell supernatant comprises direct administration at more than one site of vanishing bone disease.

[0326] Those skilled in the art will understand that the term "directly" encompasses administration of early apoptotic cells or apoptotic cell supernatant, or a composition thereof, at or near the bone being treated. Direct treatment of the bone is distinct from systemic administration, such as, but not limited to, intravenous injection or intraperitoneal infusion. In some embodiments, administration of a composition comprising an early apoptotic cell population or comprising apoptotic cell supernatant for treating vanishing bone disease does not include systemic administration.

[0327] In some embodiments, in the methods disclosed herein, direct administration comprises injecting a composition of early apoptotic cells or apoptotic cell supernatant at the affected bone site. In some embodiments, in the methods disclosed herein, direct administration comprises injecting a composition of early apoptotic cells or apoptotic cell supernatant at a site adjacent to the affected bone. In some embodiments, in the methods disclosed herein, direct administration comprises infusing a composition of early apoptotic cells or apoptotic cell supernatant at the affected bone site. In some embodiments, in the methods disclosed herein, direct administration comprises infusing a composition of early apoptotic cells or apoptotic cell supernatant at a site adjacent to the affected bone.

[0328] In some embodiments, the methods disclosed herein for treating osteoarthritis or vanishing bone disease include direct administration into a joint or at the site of an affected bone, said administration comprising infusing or injecting said early apoptotic cell population or apoptotic cell supernatant.

[0329] Early apoptotic cell colonies have been described in detail above, including but not limited to methods for preparing early apoptotic cells and compositions thereof, and therapeutically effective doses thereof. In some embodiments, the methods disclosed herein can utilize any of the above-mentioned early apoptotic cell colonies or compositions thereof, including, for example, but not limited to ApoCells, Allocetra, Allocetra-OTS, Autocetra, and Autocetra-OTS. In some embodiments, early apoptotic cells include allogeneic or autologous cells that have been irradiated. Similarly, supernatants collected from early apoptotic cell colonies or apoptotic cell colonies or apoptotic-phagocytic cell cultures have been described in detail above, including but not limited to methods for preparing these supernatants and compositions thereof, and therapeutically effective doses thereof. In some embodiments, the methods disclosed herein can utilize any of the above-mentioned supernatants or compositions thereof, including, for example, but not limited to supernatants collected from ApoCells, Allocetra, Allocetra-OTS, Autocetra, Autocetra-OTS, apoptotic cells, apoptotic cell colonies collected, and apoptotic-phagocytic cell cultures. In some embodiments, apoptotic cells used to culture the production of supernatants have been irradiated.

[0330] In some embodiments, in the methods described herein, the early apoptotic cell population includes an autologous early apoptotic cell population or an allogeneic early apoptotic cell population. In some embodiments, the early apoptotic cell population used in the methods described herein can be stable for an extended period of more than 24 hours in the early apoptotic stage. In some embodiments, in the methods described herein, the early apoptotic cell population includes an autologous early apoptotic cell population that is stable for more than 24 hours; or an allogeneic early apoptotic cell population that is stable for more than 24 hours. In some embodiments, in the methods described herein, the supernatant used is collected from an autologous early apoptotic cell population or an allogeneic early apoptotic cell population. In some embodiments, in the methods described herein, the supernatant used is collected from an apoptotic cell population or a pooled apoptotic cell population. In some embodiments, in the methods described herein, the supernatant used is collected from an apoptotic cell-phagocytic cell culture.

[0331] Methods for treating osteoarthritis or vanishing bone disease with a composition comprising an early apoptotic cell population include using cells enriched in mononuclear cells from a single source or more than one source. In some embodiments, early apoptotic cells include a confluent population of early apoptotic cells. In some embodiments, early apoptotic cells include a confluent population of early apoptotic cells, wherein the starting population of enriched mononuclear cells is obtained from a single source. In some embodiments, early apoptotic cells include a confluent population of early apoptotic cells, wherein the starting population of enriched mononuclear cells is obtained from more than one source. Cells can be collected before or after induction of apoptosis. In some embodiments, a single cell source includes cells from mismatched donors.

[0332] In some embodiments, the method of treating osteoarthritis of vanishing bone disease comprises administering a pooled early apoptotic cell population comprising an irradiated pooled early apoptotic cell population. In some embodiments, the method of treating osteoarthritis of vanishing bone disease comprises administering a supernatant collected from a pooled early apoptotic cell population comprising an irradiated pooled early apoptotic cell population, or collected from a pooled apoptotic cell population that has been irradiated.

[0333] In some embodiments, the method for treating osteoarthritis or vanishing bone disease includes the use of irradiated early apoptotic cells, wherein the cells are irradiated in a manner that will reduce the proliferation and / or activation of residual surviving cells in the apoptotic cell population. In some embodiments, in the methods disclosed herein, the cells are irradiated in a manner that reduces the percentage of non-apoptotic cells surviving in the population. In some embodiments, in the methods disclosed herein, the percentage of non-apoptotic cells surviving in the inactivated early apoptotic cell population is reduced to less than 50% of the population. In some embodiments, in the methods disclosed herein, the percentage of non-apoptotic cells surviving in the inactivated early apoptotic cell population is reduced to less than 40% of the population. In some embodiments, in the methods disclosed herein, the percentage of non-apoptotic cells surviving in the inactivated early apoptotic cell population is reduced to less than 30% of the population. In some embodiments, in the methods disclosed herein, the percentage of non-apoptotic cells surviving in the inactivated early apoptotic cell population is reduced to less than 20% of the population. In some embodiments, in the methods disclosed herein, the percentage of non-apoptotic cells surviving in the inactivated early apoptotic cell population is reduced to less than 10% of the population. In some embodiments, in the methods disclosed herein, the percentage of viable non-apoptotic cells in a population of inactivated early apoptotic cells is reduced to 0% of the population.

[0334] In another embodiment, in the methods disclosed herein, the irradiated apoptotic cells retain all of their early apoptotic, immunomodulatory and stability characteristics. In another embodiment, in the methods disclosed herein, the irradiation step uses UV radiation. In another embodiment, the irradiation step uses gamma radiation. In another embodiment, in the methods disclosed herein, the apoptotic cells comprise a reduced percentage of live non-apoptotic cells, a preparation comprising suppressed cell activation of any live non-apoptotic cells present in the apoptotic cell preparation, or a preparation comprising reduced proliferation of any live non-apoptotic cells present in the apoptotic cell preparation, or any combination thereof.

[0335] In some embodiments, in the methods disclosed herein, a cell population comprising a reduced proportion or absence of viable non-apoptotic cells, in one embodiment can provide a population without any viable / viable mononuclear early apoptotic cells.

[0336] In some embodiments, ApoCells are generated from apoptotic cells using irradiated ApoCells as shown in the examples herein (see Example 3 using a single source of autologous irradiated early apoptotic cell populations), rather than from a live proliferating cell population with cellular activity present in an apoptotic cell population.

[0337] In some embodiments, the apoptotic cells used in the methods described herein include pooled mononuclear apoptotic cell preparations. In some embodiments, the pooled mononuclear apoptotic cell preparations include mononuclear cells in an early apoptotic state, wherein the pooled mononuclear apoptotic cells include a percentage of reduced live non-apoptotic cells, a preparation in which the cell activation of any live non-apoptotic cells is inhibited, or a preparation in which the proliferation of any live non-apoptotic cells is reduced, or any combination thereof. In another embodiment, the pooled mononuclear apoptotic cells have been irradiated. In another embodiment, a pooled mononuclear apoptotic cell preparation is disclosed herein, which, in some embodiments, is derived from a white blood cell fraction (WBC) obtained from donated blood.

[0338] In some embodiments, the apoptotic cell preparation used in the methods described herein is irradiated. In another embodiment, the irradiation comprises gamma radiation or UV radiation. In yet another embodiment, the irradiated preparation has a reduced number of non-apoptotic cells compared to a non-irradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of proliferating cells compared to a non-irradiated apoptotic cell preparation. In another embodiment, the irradiated preparation has a reduced number of potentially immunocompetent cells compared to a non-irradiated apoptotic cell population. In some embodiments, the method for treating osteoarthritis or vanishing bone disease comprises directly administering a composition comprising an early apoptotic cell population comprising an irradiated early apoptotic cell population.

[0339] In some embodiments, early apoptotic cells are prepared from pooled blood collected from a single autologous donor or more than one donor, prepared and possibly stored for later use. This pooled blood pool can then be processed to produce a pooled mononuclear apoptotic cell preparation. In another embodiment, the pooled mononuclear apoptotic cell preparation ensures that a readily available supply of mononuclear apoptotic cells is available for use in treating osteoarthritis or vanishing bone disease.

[0340] In some embodiments, methods of treating osteoarthritis or vanishing bone disease comprise directly administering a composition comprising a pooled population of early apoptotic cells comprising apoptotic cells prepared from a single donor. In some embodiments, methods of treating osteoarthritis or vanishing bone disease comprise directly administering a composition comprising a pooled population of early apoptotic cells comprising apoptotic cells prepared from multiple donor mononuclear cells. In some embodiments, methods of treating osteoarthritis or vanishing bone disease comprise directly administering a composition comprising a pooled population of early apoptotic cells comprising apoptotic cells prepared from single source donor mononuclear cells or multiple donor mononuclear cells.

[0341] In some embodiments, any apoptotic supernatant disclosed herein, such as but not limited to, supernatant collected from apoptotic cells, early apoptotic cells, pooled apoptotic cells, or apoptotic cell-phagocyte cultures, can be used in methods of treating osteoarthritis or vanishing bone disease, or a combination thereof.

[0342] In some embodiments, in the method of treating osteoarthritis or vanishing bone disease, the subject is a mammalian subject. In some embodiments, in the method of treating osteoarthritis or vanishing bone disease, the subject is a human subject. In some embodiments, the human subject is a child. In some embodiments, the human subject is an adult.

[0343] The dosage of early apoptotic cells or supernatant has been described in detail above. In some embodiments, the dosage described therein can be used in the methods described herein for treating osteoarthritis or vanishing bone disease. In some embodiments, in the methods for treating osteoarthritis or vanishing bone disease, the composition of the direct administration of early apoptotic cells or supernatant comprises a single administration of early apoptotic cell colony cells or the supernatant. In some embodiments, in the methods for treating osteoarthritis or vanishing bone disease, the composition of the direct administration of early apoptotic cells or supernatant comprises more than one administration of the apoptotic cell colony or the supernatant. In some embodiments of the methods described herein, a single direct administration is administered to the affected joint. In some embodiments of the methods described herein, a single direct administration is administered to tissue as close to the affected bone as possible. In some embodiments of the methods described herein, a single direct administration is administered to adjacent to the affected joint. In some embodiments of the methods described herein, a single direct administration is administered to adjacent to the affected bone. In some embodiments of the methods described herein, more than one direct administration is administered to the affected joint. In some embodiments of the methods described herein, more than one direct administration is administered to tissue as close to the affected bone as possible. In some embodiments of the methods described herein, more than one direct administration is to an adjacent affected joint. In some embodiments of the methods described herein, more than one direct administration is to an adjacent affected bone. In some embodiments, more than one direct administration includes administration to a joint and adjacent to a joint. In some embodiments, more than one direct administration includes administration to a tissue as close as possible to the affected bone and adjacent to the affected bone.

[0344] Dosage regimens have been described in detail above. In some embodiments, methods for treating osteoarthritis or vanishing bone disease may use a dosage regimen as described above for early apoptotic cells or supernatants.

[0345] In some embodiments, in the method for treating osteoarthritis or vanishing bone disease including directly administering the composition of early apoptotic cells or supernatant, including daily or weekly administration. In some embodiments, the dosage of apoptotic cells or supernatant is a method of daily administration. In some embodiments, in the method for treating osteoarthritis or vanishing bone disease including directly administering the composition of early apoptotic cells or supernatant, including weekly administration of apoptotic cells or supernatant. In some embodiments, the dosage of apoptotic cells or supernatant is administered every half week (twice a week). In some embodiments, the dosage of apoptotic cells or supernatant is administered every two weeks (bi-weekly) (every two weeks). In some embodiments, the dosage of apoptotic cells or supernatant is administered monthly. In some embodiments, the dosage of apoptotic cells or supernatant is administered in an irregular scheme, such as daily administration within a given time period, followed by half a week, or weekly, or two weeks, or monthly administration, or a combination thereof.

[0346] The dosage of early apoptotic cells or supernatant has been described in detail above. As mentioned above, in some embodiments, the method of use for treating osteoarthritis or vanishing bone disease may use a certain dosage of early apoptotic cells or supernatant.

[0347] In some embodiments, a method of treating osteoarthritis or vanishing bone disease comprises directly administering a 6 -210×10 6 In some embodiments, in a method for treating osteoarthritis or vanishing bone disease, comprising directly administering a supernatant containing about 1×10 6 -1×10 9 In some embodiments, administering about 1×10 6 -1×10 8 In some embodiments, administration of about 1×10 6 -1×10 7 In some embodiments, administration of about 1×10 7 -1×10 9 In some embodiments, administration of about 1×10 7 -1×10 8 In some embodiments, administration of about 1×10 8 -1×10 9 In some embodiments, administration of about 1×10 6 In some embodiments, administration of about 1×10 7In some embodiments, administration of about 1×10 8 In some embodiments, administration of about 1×10 9 The dose of apoptotic cells.

[0348] In some embodiments, about 140×10 6 -210×10 6 In some embodiments, about 100×10 6 -140×10 6 In some embodiments, about 10-100×10 6 In some embodiments, about 20×10 6 In some embodiments, about 30×10 6 In some embodiments, about 40×10 6 In some embodiments, about 50×10 6 In some embodiments, 60×10 6 In some embodiments, about 60×10 6 In some embodiments, about 70×10 6 In some embodiments, about 80×10 6 In some embodiments, about 90×10 6 In some embodiments, about 1-15×10 7 In some embodiments, about 10×10 7 In some embodiments, about 11×10 7 In some embodiments, about 12×10 7 In some embodiments, about 13×10 7 In some embodiments, about 14×10 7 In some embodiments, about 15×10 7 In some embodiments, 1×10 6 In some embodiments, 10×10 6 In another embodiment, 10×107 In another embodiment, 10×10 8 In another embodiment, 10×10 9 In another embodiment, 10×10 10 In another embodiment, 10×10 11 In another embodiment, 10×10 12 In another embodiment, 10×10 5 In another embodiment, 10×10 4 In another embodiment, 10×10 3 In another embodiment, 10×10 2 The dose of apoptotic cells.

[0349] In some embodiments, the method of treating osteoarthritis or vanishing bone disease or a combination thereof uses a high dose of apoptotic cells. In some embodiments, depending on the size of the joint, the method of treating osteoarthritis or vanishing bone disease or a combination thereof uses a low dose of apoptotic cells. In some embodiments, 10×10 6 In some embodiments, 15×10 6 In some embodiments, 20×10 6 In some embodiments, 25×10 6 In some embodiments, 30×10 6 In some embodiments, 35×10 6 In some embodiments, 50×10 6 In some embodiments, 100×10 6 In some embodiments, 150×10 6 In some embodiments, 200×10 6 In another embodiment, 210×10 6 In another embodiment, 70×10 6 In another embodiment, 140×10 6In another embodiment, 35-210×10 6 The dose of apoptotic cells.

[0350] In some embodiments, in a method of treating osteoarthritis or vanishing bone disease or a combination thereof, the method comprises directly administering a composition disclosed herein comprising a supernatant comprising from about 100×10 6 -210×10 6 In some embodiments, the method of treating osteoarthritis or vanishing bone disease comprises directly administering a supernatant containing about 1×10 6 -1×10 9 In some embodiments, administering a supernatant collected from about 1×10 6 -1×10 8 In some embodiments, administration of supernatant collected from about 1×10 6 -1×10 7 In some embodiments, administration of supernatant collected from about 1×10 7 -1×10 9 In some embodiments, administration of supernatant collected from about 1×10 7 -1×10 8 In some embodiments, administration of supernatant collected from about 1×10 8 -1×10 9 In some embodiments, administration of supernatant collected from about 1×10 6 In some embodiments, administration of supernatant collected from about 1×10 7 In some embodiments, administration of supernatant collected from about 1×10 8 In some embodiments, administration of supernatant collected from about 1×10 9 The supernatant of apoptotic cells was collected.

[0351] In some embodiments, administering from about 140×10 6 -210×10 6 In some embodiments, the supernatant collected from about 100×10 6 -140×10 6 In some embodiments, the supernatant collected from about 10-100×10 6 In some embodiments, administration of supernatant collected from about 20×10 6In some embodiments, administration of supernatant collected from about 30×10 6 In some embodiments, administration of supernatant collected from about 40×10 6 In some embodiments, administration of supernatant collected from about 50×10 6 In some embodiments, administration of supernatant collected from about 60×10 6 In some embodiments, administration of supernatant collected from about 60×10 6 In some embodiments, administration of supernatant collected from about 70×10 6 In some embodiments, administration of supernatant collected from about 80×10 6 In some embodiments, administration of supernatant collected from about 90×10 6 In some embodiments, administering from about 1-15×10 7 In some embodiments, administration of supernatant collected from about 10×10 7 In some embodiments, administration of supernatant collected from about 11×10 7 In some embodiments, administration of supernatant collected from about 12×10 7 In some embodiments, administration of supernatant collected from about 13×10 7 In some embodiments, administration of supernatant collected from about 14×10 7 In some embodiments, administration of supernatant collected from about 15×10 7 In some embodiments, supernatant collected from 1×10 6 In some embodiments, supernatant collected from 10×10 6 In another embodiment, supernatant collected from 10×10 7 In another embodiment, supernatant collected from 10×10 8 In another embodiment, supernatant collected from 10×10 9 In another embodiment, supernatant collected from 10×10 10 In another embodiment, supernatant collected from 10×10 11 In another embodiment, supernatant collected from 10×10 12In another embodiment, supernatant collected from 10×10 5 In another embodiment, supernatant collected from 10×10 4 In another embodiment, supernatant collected from 10×10 3 In another embodiment, supernatant collected from 10×10 2 The supernatant of apoptotic cells was collected.

[0352] In some embodiments, the method of treating osteoarthritis or vanishing bone disease or a combination thereof administers a high dose of supernatant collected from apoptotic cells or apoptotic-phagocytic cell cultures. In some embodiments, depending on the size of the joint, the method of treating osteoarthritis or vanishing bone disease or a combination thereof administers a low dose of supernatant collected from apoptotic cells or apoptotic-phagocytic cell cultures. In some embodiments, administering a supernatant collected from 10×10 6 In some embodiments, supernatant collected from 15×10 6 In some embodiments, supernatant collected from 20×10 6 In some embodiments, supernatant collected from 25×10 6 In some embodiments, supernatant collected from 30×10 6 In some embodiments, supernatant collected from 35×10 6 In some embodiments, supernatant collected from 50×10 6 In some embodiments, supernatant collected from 100×10 6 In some embodiments, supernatant collected from 150×10 6 In some embodiments, supernatant collected from 200×10 6 In another embodiment, supernatant collected from 210×10 6 In another embodiment, supernatant collected from 70×10 6 In another embodiment, supernatant collected from 140×10 6 In another embodiment, the supernatant collected from 35-210×10 6 The supernatant of apoptotic cells was collected.

[0353] In some embodiments of the methods described herein, the number of early apoptotic cells is ±5%, ±10%, ±15%, or ±20% of the number of cells. In some embodiments of the methods described herein, the number of apoptotic cells is ±5%, ±10%, ±15%, or ±20% of the number of cells. For example, but not limited to, each dose administered contains about 50×10 6 ±5% to 200×10 6 ±5% early apoptotic cells / kg subject, or 50×10 6 ±10% to 200×10 6 ±10% early apoptotic cells / kg subject, or 50×10 6 ±15% to 200×10 6 ±15% early apoptotic cells / kg subject, or 50×10 6 ±20% to 200×10 6 ±20% early apoptotic cells / kg subject. Similarly, supernatant can be collected from cultures of apoptotic cells in the range of ±5%, ±10%, ±15% or ±20% of the number of cells. Example

[0354] Example 1: Generation of apoptotic cells

[0355] Objective: To generate early apoptotic cells, including irradiated early apoptotic cells.

[0356] Methods: Methods for preparing early apoptotic cell populations are well documented in International Publication No. WO 2014 / 087408 and U.S. Application Publication No. US2015 / 0275175-A1, e.g., see the “Methods” section preceding the “Examples” of “Early apoptotic cell population Preparation” and “Generation of apoptotic cells” (paragraphs

[0223] to

[0288] ) and Examples 11, 12, 13, and 14, which are incorporated herein by reference in their entireties).

[0357] Figure 1The flowchart presented in provides an overview of one embodiment of the steps used in the method for producing an early apoptotic cell population, wherein an anticoagulant is included in the thawing and apoptosis induction steps. As described in detail in Example 14 of International Publication No. WO2014 / 087408 and U.S. Application Publication No. US US-2015-0275175-A1, an early apoptotic cell population is prepared, wherein an anticoagulant is added during freezing, or during incubation, or during freezing and incubation. The anticoagulant used is citrate-dextrose, and NIH Formula A (ACD Formula A) is supplemented with 10U / ml heparin, with a final concentration of 5% ACD and 0.5U / ml heparin of the total volume.

[0358] In certain embodiments, the cell populations used as the starting populations can be different, yet the methods described herein for producing early apoptotic cells result in the same quality and characteristics of the final product. In some embodiments, the cells used to produce early apoptotic cells can be autologous, allogeneic from a donor, or allogeneic from a blood bank.

[0359] Briefly: cells were collected and then frozen, with 5% anticoagulant citrate dextrose formula A and 10 U / ml heparin (ACDhep) added to the freezing medium. Thawing, incubation in apoptosis-inducing medium containing 5% ACDhep and final product preparation were performed in a closed system.

[0360] In each experiment, apoptosis and viability analysis, titer determination and cell colony characterization were carried out. In order to establish the consistency of early apoptotic cell product production, the final product (FP) of the initial batch of apoptotic cells was stored at 2-8 ° C and checked at t0, t24h, t48h and t72h. At each point, apoptosis analysis, short titer determination (applicant CD14+ frozen cells), trypan blue measurement and cell colony characterization were carried out. FP was tested for cell counting, to assess the average cell loss during storage and apoptosis and viability analysis.

[0361] The Methods section referenced above and Example 11 of International Publication No. WO 2014 / 087408 and US Application Publication No. US-2015-0275175-A1 provide details of other embodiments for preparing apoptotic cell populations in the absence of anticoagulants and are incorporated herein in their entirety.

[0362] In some cases, after early apoptotic cells are prepared (after induction of apoptosis), in other words, after Figure 1 After the final step shown, they are irradiated. In other embodiments, irradiation can occur at an earlier step in the procedure to produce irradiated early apoptotic cells.

[0363] Methods for preparing irradiated apoptotic cells: A similar method is used to prepare an inactivated apoptotic cell population, wherein a mononuclear early apoptotic cell population from a single source or more than one source comprises a reduced percentage of non-quiescent non-apoptotic cells, or a cell population in which the cell activity of any live non-apoptotic cell is inhibited, or a cell population in which the proliferation of any live non-apoptotic cell is reduced, or any combination thereof.

[0364] Briefly, enriched mononuclear cell fractions are collected from healthy, qualified donors by a leukocyte apheresis procedure. After apheresis is completed, the cells are washed and resuspended in freezing medium containing 5% anticoagulant citrate dextrose solution-Formula A (ACD-A) and 0.5U / ml heparin. The cells are then gradually frozen and transferred to liquid nitrogen for long-term storage. In some embodiments, more than one fraction from different donors or the same donor is pooled.

[0365] In order to prepare irradiated ApoCells (early apoptotic cells), the cryopreserved cells were thawed, washed and resuspended with an apoptosis-inducing medium containing 5% ACD-A, 0.5U / ml heparin sodium and 50μg / ml methylprednisolone. The cells were then incubated at 37°C for 6 hours in 5% CO2. At the end of the incubation, the cells were collected, washed and resuspended in Hartmann solution using a cell processing system (Fresenius Kabi, Germany). In some embodiments, the collected apoptotic cell fractions are pooled to produce pooled apoptotic cell fractions. After manufacturing was completed (inducing apoptosis), ApoCells were irradiated with 4000cGy using a g-camera at the radiotherapy unit Hadassah Ein Kerem. By flow cytometry, the apoptosis and survival of ApoCells (≥40% and ≤15%, respectively) were determined using annexin V and PI (MBL, MA, USA) staining. The results were analyzed using FCSexpress software. In some embodiments, after irradiation, a single source of irradiated apoptotic cells is pooled, where the source is the same or a different donor. In other embodiments, more than one single source or more than one different source of apoptotic cells are pooled before irradiation.

[0366] Such irradiated ApoCell populations are believed to include early apoptotic cells, wherein any surviving cells present have suppressed cell activity and reduced or absent proliferation capacity. In some cases, the ApoCell population is devoid of surviving non-apoptotic cells.

[0367] result:

[0368] The stability of the FP produced when an anticoagulant was included during freezing and incubation (apoptosis induction) and then stored at 2-8°C is shown in Table 1 below.

[0369] Table 1: Cell counts* - performed using a MICROS 60 hematology analyzer.

[0370] FP time point <![CDATA[Cell concentration (x10 6 cells / ml)]]> Cell loss% t0 20.8 NA t24h 20.0 -3.85 t48h 20.0 -3.85 t72h 19.7 -5.3

[0371] *Represents the results of 6 (six) experiments.

[0372] When cells were produced without anticoagulants in the induction medium, the cells were stable for 24 hours and less stable thereafter. The use of anticoagulants unexpectedly extended the stability of the apoptotic cell population for at least 72 hours, as shown in Table 1.

[0373] Table 2: Trypan blue measurement

[0374] FP time point Trypan blue positive cells (%) t0 3.0 t24h 5.9 t48h 5.2 t72h 6.5

[0375] The results in Table 2 show that the viability of FP remained high for at least 72 hours.

[0376] Table 3: Cell apoptosis analysis by flow cytometry (AnPI staining)

[0377]

[0378] The results in Table 3 show that the percentage of apoptotic cells relative to necrotic cells remained constant over an extended period of at least 72 hours after cell preparation, as did the percentage of early apoptotic cells.

[0379] The data in Table 3 confirm that most of the cells in the generated population are in early apoptosis, wherein the percentage of cells in early apoptosis in the population (An+PI-) is greater than 50%, and in some cases greater than 60%. The generated cell population contains a minimum percentage of late apoptotic cells or dead cells (less than or equal to 6%). See also Table 5 below.

[0380] The inclusion of an anticoagulant both at the time of freezing and during the induction of apoptosis resulted in the most consistently high yield of stable early apoptotic cells (mean yield of early apoptotic cells was 61.3% ± 2.6% (An + PI-) vs. 48.4% ± 5.0%, where 100% yield is based on the number of cells at the time of freezing). This high yield was maintained even after storage at 2-8°C for 72 hours.

[0381] Next, a comparison was made between the inclusion of anticoagulants upon freezing or thawing or both, where percent recovery (%) and stability were measured. Anticoagulants were included in the apoptosis incubation mix for all populations. Table 4 presents the results of these studies.

[0382] Table 4: Comparison of the yield and stability of the final product (FP) made from cells harvested with ("+") or without ("-") anticoagulant added during freezing ("F") and thawing ("Tha").

[0383]

[0384] Additional population analysis comparisons of early apoptotic cell populations (cell batches) prepared with and without the addition of anticoagulants showed consistency of these results.

[0385] Table 5: Comparison of cell population analysis between batches prepared with and without anticoagulants

[0386]

[0387]

[0388] The percentage (yield) of final product cells in the presence or absence of anticoagulants. Similar to the results presented in Table 1 above, the data given in Table 4 show that the early apoptotic cells manufactured from cells frozen in the presence of anticoagulants have a beneficial effect on the average yield of fresh final products (FP t0) compared to cells frozen in the absence of anticoagulants. When anticoagulants are used only when freezing (61.3% ± 2.6% vs. 48.4% ± 5.0%), or when both freezing and thawing (56.5% ± 5.2% vs. 48.4% ± 5.0%), a beneficial effect is observed. When anticoagulants are used only when thawing, the beneficial effect is less significant (44.0% ± 8.5% vs. 48.4% ± 5.0%). These are non-high triglyceride samples.

[0389] Effect of anticoagulants on aggregation. Cell aggregation was not observed in these 3 non-high triglyceride samples or 21 additional samples (data not shown). However, in the other 41 non-high triglyceride samples made without anticoagulants (data not shown), mild aggregation was observed in 10 samples (24.4%), and severe aggregation was observed in 5 samples (12.2%); Therefore, anticoagulants completely avoided cell aggregation.

[0390] Effect of Anticoagulant on Stability. Fresh FP manufactured with or without anticoagulant was stored at 2–8°C for 24 h to determine if the addition of ACDhep during the manufacturing procedure would compromise the stability of the FP. Cells were sampled after 24 h of storage and the yield was calculated. Similar to the results for extended time periods (up to 72 h) shown in Table 1, Table 4 shows that the beneficial effects were maintained and observed when anticoagulants were used only at freezing (59.8% ± 2.1% vs. 47.5% ± 4.7%), or when anticoagulants were used both at freezing and thawing (56.4% ± 5.3% vs. 47.5% ± 4.7%). The beneficial effects were less pronounced when anticoagulants were added only at thawing (42.4% ± 6.1% vs. 47.5% ± 4.7%). These were non-high triglyceride samples. These results show that FP had minimal cell loss after 24 h of storage among all treatments, with a significant advantage for cells treated with anticoagulants during both freezing and thawing. The mean loss of cells treated with anticoagulants during freezing only was 2.3% ± 3.2% compared to 1.9% ± 3.3% without anticoagulants, 3.0% ± 4.7% during thawing only compared to 1.9% ± 3.3% without anticoagulants, and 0.2% ± 0.4% when cells were frozen and thawed in the presence of ACDhep compared to 1.9% ± 3.3% without anticoagulants. In conclusion, the beneficial effects of anticoagulants on yield were maintained for at least 24 hours.

[0391] The characteristics of representative cell populations of FP are shown in Table 6 below.

[0392] Table 6: Characterization of fresh (t0) FP cell populations made from cells collected with ("+") or without ("-") addition of anticoagulant during the freezing ("F") and thawing ("Tha") procedures*

[0393]

[0394] *Apoptosis induction was performed for all batches using medium containing anticoagulant.

[0395] The results in Table 6 show the cell characteristics of the final product (FP) manufactured with or without anticoagulants during freezing and thawing. Batch samples were taken, stained for mononuclear markers, and analyzed by flow cytometry to determine the cell distribution in each sample and to examine whether the addition of anticoagulants affected the cell population. As shown in Table 5, no significant differences were detected in the cell populations manufactured with or without anticoagulants during freezing or thawing. The mean T cell population (CD3+ cells) in fresh FP was 62.3%±1.2% between treatments, compared to 62.9%±1.1% before freezing; the mean B cell population (CD19+ cells) was 8.3%±2.5% between treatments, compared to 3.1%±0.8% before freezing; the mean natural killer cell population (CD56+ cells) was 9.5%±0.7% between treatments, compared to 12.9%±0.5% before freezing; the mean mononuclear cell population (CD14+ cells) was 13.8%±0.5% between treatments, compared to 17.5%±0.3% before freezing; and the mean granulocyte population (CD15+ cells) was 0.0% in fresh FP, compared to 0.35%±0.2% when frozen.

[0396] The potency of the early apoptotic population was also examined.

[0397] Table 7: Potency analysis of fresh (t0) FP made from cells with added ("+") or without added ("-") anticoagulant during the freezing ("F") and thawing ("Tha") procedures.

[0398]

[0399] The results presented in Table 7 are from potency assays, which were performed to determine the ability of each final product to enhance the tolerogenic state of immature dendritic cells (iDC) after stimulation with (LPS). The tolerogenic effect was determined by assessing the downregulation of the expression of costimulatory molecules HLA-DR and CD86 on iDC after different treatments that interacted with early apoptotic cell populations and resulted in LPS upregulation. DCsign+ cells were analyzed. The results represent the percentage delay of maturation relative to LPS-induced maturation after interaction with early apoptotic cell populations and after the addition of lipopolysaccharide. The experiment tested the potency of fresh FP (t0) manufactured with or without anticoagulants. The results presented in Table 7 show that apoptotic cells manufactured with or without anticoagulants enhanced the tolerogenic effect of two costimulatory markers in a dose-dependent manner.

[0400] The early apoptotic cells produced herein are from non-high triglyceride samples. Even in the case of high triglyceride donor plasma, this consistent high yield of stable early apoptotic cells with high yield is produced (see, e.g., Examples 12 and 13 of International Publication No. WO 2014 / 087408 and U.S. Application Publication No. US US-2015-0275175-A1). It should be noted that no anticoagulant is added to the PBS medium used to prepare the final early apoptotic cell dose for infusion.

[0401] Summarize:

[0402] The purpose of this study is to produce a stable, high-yield early apoptotic cell population. The rationale for using anticoagulants is that aggregates are first observed in patients with high triglycerides, but then observed in a significant proportion of other patients. A consideration here is that the use of anticoagulants to prevent apoptosis is disclosed in U.S. Patent No. US 6,489,311.

[0403] In short, when the addition of anticoagulants was added, the number of cells collected in the final product (yield) was significantly increased by at least 10%-20% with minimal impact on the composition, viability, stability and apoptotic properties of the cells. In this study, the yield was shown to increase by up to 13%, which means that under controlled conditions the yield increased by 26.8%, but under actual GMP conditions, the yield increased by up to 33%, and then a greater increase in the number of cells could be produced in a single collection. This effect is crucial because it can avoid the need for a second apheresis of the donor.

[0404] This effect was surprising as the expected effect was expected to be the dissolution of mild aggregates. It was hypothesized that thawing the cells in the presence of an anticoagulant would reduce the number of aggregates. When these aggregates form, they ultimately lead to significant cell loss. Cells collected and frozen in the absence of an anticoagulant showed the formation of aggregates when thawed immediately after washing. In addition, high levels of aggregates were also detected in cells that were frozen in the absence of an anticoagulant and resuspended in medium containing an anticoagulant. No aggregates were observed in cells that were both frozen and resuspended in medium containing an anticoagulant. Taken together, it was concluded that the addition of an anticoagulant during freezing and apoptosis induction is very important and does not appear to negatively affect the induction of early apoptosis in the cell population.

[0405] The recovery of early apoptotic cells was further tested, for example, after 24 h of storage at 2–8°C for stability purposes, during which an average cell loss of 3%–4.7% was measured, and for cells frozen and thawed in medium containing an anticoagulant, the results were favorable (0.2% ± 0.4% cell loss after 24 h of FP storage), regardless of manufacturing conditions, indicating that the addition of an anticoagulant during freezing and thawing is critical, but once finalized, the early apoptotic cell population is stable. Extended time point studies showed this stability to at least 72 h.

[0406] The addition of anticoagulants during the freezing and / or thawing stages did not significantly affect apoptosis and viability and cell composition of the FP product. The values ​​measured from the various characteristics were similar, indicating that ACDhep did not alter early apoptotic cell characteristics and that the final product met the acceptance criteria of ≥40% apoptotic cells.

[0407] The assay used to test the potency of apoptotic cells is based on immature dendritic cells (iDCs), which are DCs characterized by functions such as phagocytosis, antigen presentation, and cytokine production.

[0408] HLA-DR (class II MHC) membrane molecules and co-stimulatory molecules CD86 were selected as markers for the tolerogenic effect of antigen presenting cells (APC). Using flow cytometry, changes in HLA-DR and CD86 expression on iDC were measured after stimulation with LPS, and in the presence of an early apoptotic cell colony made with or without an anticoagulant and stimulated with LPS. The early apoptotic cell colony was provided to DC with an increasing ratio of 1:2, 1:4 and 1:8 iDC: early apoptotic cell colonies. As shown in Table 6, it was shown that the early apoptotic cell colony enhanced the tolerogenic effect of the stimulated DC in a dose-dependent manner, wherein the early apoptotic cell colony made with an anticoagulant when frozen and apoptosis induced, the result was slightly better.

[0409] In summary, we conclude that the addition of anticoagulants to the freezing and apoptosis media is important to improve cell recovery and avoid significant cell loss due to aggregation and, in many cases, avoid a second round of apheresis of the donor. All cells were shown to meet the acceptance criteria for a validated FP, indicating that the addition of anticoagulants does not impair the FP.

[0410] Example 2: Preparation and use of pooled apoptotic cells

[0411] Objective: To generate irradiated multi-donor single apoptotic cell infusions (pooled mononuclear irradiated apoptotic cell preparations).

[0412] Method: Apoptotic cells were prepared as described in Example 1 above, except that in the current experiment, preparations were performed simultaneously from more than one (4) donors. After preparation from 4 donors, the cell preparations were pooled in the last step (before irradiation), immediately irradiated, and ready for use. The irradiation was 25 Gy.

[0413] Results and Summary:

[0414] Analysis in a GvHD mouse model showed that a single infusion of irradiated apoptotic cells from more than one donor successfully and significantly improved life expectancy in the GvHD mouse model. (Data not shown)

[0415] Example 3: Preparation of apoptotic cell supernatant

[0416] Objective: To obtain supernatants from early apoptotic cells and monocytes / macrophages / dendritic cells.

[0417] Methods: Preparation from apoptotic cells: CD14+ monocytes and other mononuclear cell-enriched leukocytes were cultured and triggered to undergo apoptosis. The number of apoptotic cells ranged from 1 million to 100 million cells per well in a 12-well plate. In some cases, 8 million cells were cultured per well. After 24, 36, and 48 hours of incubation, the cells were centrifuged (290 g, 4 degrees Celsius, 10 minutes). The supernatant was collected and frozen in aliquots at -80 degrees Celsius until use.

[0418] Methods: Preparation from monocytes / macrophages / dendritic cells and apoptotic cells: CD14+ monocytes were cultured with apoptotic cells prepared as above at a ratio of 1:16 for 24 hours, 36 hours or 48 hours. The number of monocytes was: 0.5 million cells per well in a 12-well plate, and the number of apoptotic cells was: 8 million cells per well in a 12-well plate. After incubation for 24 hours, the cells were centrifuged (290g, 4 degrees Celsius, 10 minutes). The supernatant was collected and frozen in aliquots at -80 degrees Celsius until use. Similar procedures can be performed with different ratios of mononuclear cells: apoptotic cells and / or using cells derived from other sources of monocytes, such as different types of macrophages, including M1 / M2 and dendritic cells.

[0419] Results: The apoptotic supernatants prepared from apoptotic mononuclear cells or from monocytes / macrophages / dendritic cells co-cultured with apoptotic mononuclear cells herein are able to downregulate proinflammatory cytokines under cytokine storm conditions (data not shown). In certain embodiments, these apoptotic supernatants can be effectively used in the methods disclosed herein for treating osteoarthritis.

[0420] Example 4: Treatment of osteoarthritis using apoptotic cells

[0421] Objective: To treat osteoarthritis in subjects who have not responded to other therapies.

[0422] Method: Early apoptotic cells were prepared as described in Example 1 above.

[0423] The subject treated was a 70-year-old female with hypothyroidism. She had been experiencing an inflammatory and erosive process in her right shoulder for 4 years. In 2015, she experienced swelling of the right upper extremity and limited range of motion of the right shoulder in the absence of a known injury. X-rays showed complete destruction of the humeral head, MRI showed a marked inflammatory reaction, and marked elevations in erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) were observed. No serum autoantibodies or bacterial growth were detected on needle aspiration of the joint. After systemic steroid therapy, the ESR and CRP decreased, but there was no significant clinical improvement. After an uneventful total reverse shoulder arthroplasty, the swelling, ESR, and elevated CRP gradually resolved. Two years later, the patient returned with swelling in the shoulder and a marked elevation in CRP. Repeated aspirations, debridements, and finally removal of the prosthesis were performed, followed by long-term antibiotic therapy for the possibility of infection. The swelling and CRP levels improved only with continued shoulder drainage. She was hospitalized for more than 9 months last year. The ethics committee authorized the use of her own irradiated apoptotic cells for compassionate care.

[0424] Irradiated autologous early apoptotic cells (at Figure 2 Patients underwent leukapheresis to obtain the starting material from which irradiated early apoptotic cells were prepared. Irradiated early apoptotic cells (100×10 6 ) were administered intra-articularly in the right shoulder joint for 5 consecutive weeks. Specifically, each dose of cells administered contained 100×10 6 ± 20% of irradiated early apoptotic cells. Administered by intra-articular infusion.

[0425] Measurement of cytokines and chemokines present in synovial fluid was performed by Luminex MAGPIX system (Luminex USA) using Milliplex software (Merck).

[0426] result:

[0427] After weekly intra-articular infusion of apoptotic cells for 5 weeks, the patient improved significantly, with a significant reduction in shoulder swelling, redness, and tightness within 6 weeks. Fluid discharge from the shoulder decreased from 150–250 ml / day to <60 ml / day. CRP decreased from 7.34 before treatment to 0.49 (normal range ≤0.5). Synovial fluid samples were collected before and weekly during / after treatment. Measurement of proinflammatory cytokines / chemokines associated with monocyte, macrophage, dendritic cell, osteoclast, neutrophil, and T cell activation showed downregulation of IL-6, IL-8, IL-1β, IL-2, IL-15, IL-22, MIP-1β, IL-9, and TNFα after 2 injections of irradiated early apoptotic cells ( Figure 2 ). Figure 2 The most notable ones were down-regulation of IL-22 (dysregulation of synovial tissue wound healing), IL-8 (neutrophil chemotactic factor), IL-6 (innate immunity), IL-9 (prevention of apoptosis), and MIP-1-β (chronic inflammation).

[0428] There were no significant changes in blood biochemistry and CBC. An infection caused by the drain that occurred 3 weeks after the end of treatment was treated with antibiotics, debridement, and drain replacement. Due to low fluid output, the drain was removed 3 months after treatment and no longer required hospitalization. At the 6-month follow-up, CRP remained low.

[0429] The effects observed in the first five weeks of treatment of the patient were maintained for nine months, during which time she stayed at home and remained stable after the intervention. Surprisingly and unexpectedly, although IL-6 and possibly IL-8 are cytokines that have been described in vanishing bone disease and their elevation can be predicted, this is the first time that high IL-22 has been described, which is associated with dysregulated synovial healing. Its elevation and the effect of early apoptotic cell therapy were unexpected. The same can be said for IL-9, which can avoid apoptosis of inflammatory cells, thereby maintaining chronic inflammation, while the downregulation of IL-9 may trigger activation-induced cell death and terminate the immune response. In addition, surprisingly, TNF did not rise at all. In this regard, anti-cytokines such as anti-TNF and even anti-IL-6 are not a good choice, and only apoptotic cells have this overall effect.

[0430] Summarize:

[0431] HLA-matched apoptotic cell infusion has previously been reported to be safe and beneficial in preventing graft-versus-host disease (Mevorach D, Zuckerman T, Reiner I, et al. (2014) "Single infusion of donor mononuclear early apoptotic cells as prophylaxis for graft-versus-host disease in myeloablative HLA-matched allogeneic bone marrow transplantation: a phase I / IIa clinical trial." Biol Blood Marrow Transplant., 20: 58-65), but this is the first time that autologous early apoptotic cells prepared specifically as described in this article have been used for intra-articular infusion.

[0432] The early apoptotic cell preparation prepared for the treatment of the patient is autologous, however, those skilled in the art will appreciate that it is also possible to administer a non-autologous preparation of these early apoptotic cells to achieve the same results. Early apoptotic cells can be obtained from a third party subject, or can be a third party pooled apoptotic cell preparation (allogeneic), which is then subjected to a radiation step prior to administration and successfully treats osteoarthritis.

[0433] The mechanism of action of this treatment is believed to be related to signaling in monocytes, macrophages, dendritic cells, and osteoclasts (Trahtemberg U, Mevorach D. (2017) "Apoptotic cells induced signaling for immune homeostasis in macrophages and dendritic cells." Front Immunol., 8: 1356).

[0434] Considering the conclusion of Grey et al. (supra) that if the selected route of administration does not deliver apoptotic cells to the spleen so that the apoptotic cells can interact with B cells, protection is unlikely to be induced, it is very unexpected that intra-articular infusion for non-autoimmune conditions (e.g., erosive osteoarthritis) would be successful because apoptotic cells administered by intra-articular infusion cannot enter the spleen or immune organs. Therefore, it is surprising and unexpected that intra-articular infusion terminates the increased release of pro-inflammatory and anti-inflammatory cytokines / chemokines in the synovial fluid. In particular, high IL-22 associated with dysregulated synovial healing and downregulation of IL-9, which can avoid apoptosis of inflammatory cells and thus maintain chronic inflammation, which downregulation can trigger the termination of chronic inflammation.

[0435] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0436] Throughout this application, various embodiments of the present invention can be presented in the form of ranges. It should be understood that the description of range form is only for convenience and brevity, and should not be interpreted as a rigid limitation to the scope of the present invention. Accordingly, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, and single numerical values ​​such as 1, 2, 3, 4, 5 and 6 within the range. Regardless of the width of the scope, this applies.

[0437] Whenever a numerical range is indicated herein, it is intended to include any cited numerical value (fractional or integer) within the indicated range. The terms "ranging / ranges between" a first indicated number and a second indicated number "between" and "ranging / ranges from" a first indicated number "to" a second indicated number are used interchangeably herein and are intended to include the first indicated number and the second indicated number and all fractions and integers therebetween.

[0438] While certain features have been illustrated and described herein, numerous modifications, substitutions, variations, and equivalents will now occur to those of ordinary skill in the art. It should therefore be understood that the appended claims are intended to cover all such modifications and variations that fall within the true spirit of the methods of use presented herein.

Claims

1. Use of a pharmaceutical composition comprising an enriched early apoptotic cell population of mononuclear cells for the manufacture of a medicament for treating osteoarthritis in a subject in need thereof, wherein the enriched early apoptotic cell population of mononuclear cells comprises more than 40% annexin-V and less than 15% PI positive cells and is irradiated after induction of apoptosis, wherein the pharmaceutical composition is formulated for direct administration into the joints of the subject.

2. The use according to claim 1, wherein the early apoptotic cell population is formulated for infusion or injection into the joint.

3. The use according to claim 1, wherein the early apoptotic cell population comprises (a) autologous early apoptotic cell population; or (b) Allogeneic early apoptotic cell population. The use according to claim 1 , wherein the early apoptotic cell population comprises a pooled early apoptotic cell population.

5. The use according to claim 4, wherein the pooled early apoptotic cell population comprises apoptotic cells prepared from single donor mononuclear cells or from multiple donor mononuclear cells.

6. The use according to claim 1, wherein the subject is a human subject.

7. The use according to claim 1, wherein the dose for each administration comprises 1×10 6 ±20% to 1×10 9 ±20% early apoptotic cells / kg subject.

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