WO2016075542A1

WO2016075542A1 - Methods for extended storage of activated leukocyte compositions

Info

Publication number: WO2016075542A1
Application number: PCT/IB2015/002285
Authority: WO
Inventors: Adi Zuloff-Shani; Shik PAVEL; Peer IDAN
Original assignee: Macrocure, Ltd.
Priority date: 2014-11-11
Filing date: 2015-11-10
Publication date: 2016-05-19

Abstract

Disclosed are storage solutions for activated leukocyte compositions. More particularly, disclosed are storage solutions that allow activated leukocyte compositions to be stored for an extended period of time at reduced temperatures, while retaining leukocyte stability and activity over time.

Description

METHODS FOR EXTENDED STORAGE OF ACTIVATED LEUKOCYTE COMPOSITIONS

This application is entitled to and claims the benefit of priority from United States Provisional Application No. 62/078, 165 filed November 1 1 , 2014, the contents of which are expressly incorporated herein by reference.

BACKGROUND

The wound healing process involves participation of white blood cells, also known as leukocytes. Leukocytes include lymphocytes, granulocytes and monocytes. Three common types of lymphocytes are T-cells, B-cells and natural killer cells. T-cells and B-cells play important roles in the recognition of antigens in the body (Parkin, 2001 ). Natural killer (NK) cells identify infected cells by alterations in the levels of the major histocompatability complex (MHC), and destroy the infected cells (Moretta, 2008). The participation of lymphocytes in the healing process is largely associated with their production of cytokines and growth factors (Keen, 2008). A new class of gamma-delta- T cells has been described in the skin (Jameson, 2002; Havran, 2005). Among the different types of granulocytes are neutrophils, basophils, and eosinophils. Monocytes differentiate into macrophages, which are responsible for destruction of tissue debris or invading foreign substances. Macrophages also produce molecules that control inflammation and repair (Riches, 1996). All of these cells types contribute to the process of wound healing in mammals.

The process of wound healing occurs in three overlapping phases. (Li, 2007; Broughton, 2006; Tsirogianni, 2006; Singer, 1999; Martin, 1997). The first phase is the inflammatory phase. It is characterized by recruitment of neutrophils, followed by monocytes to the wound site, where they kill and phagocytize bacteria (Agaiby, 1999).

The second wound healing phase which is known as the proliferative phase, involves formation of new granulation tissue. Fibroblasts proliferate and migrate into the wound space and synthesize collagen and other components of extracellular matrix (Greiling, 1997). At the same time, angiogenesis occurs, providing nutrients and oxygen to the metabolically active new granulation tissue (Tonnesen,

2000). Keratinocytes from the intact epidermis start to migrate over the provisional matrix and begin to proliferate, leading the way for new epithelial tissue (Kim, 1992).

Remodeling is the third and final phase in wound healing. It is characterized by fibroblast differentiation into myofibroblasts, which contract and bring the wound edges closer together (Tomasek, 2002). Remodeling of the collagen fibers by degradation and re-synthesis allows the wound to gain strength by re-orientation of the collagen fibers (a process tightly controlled by growth factors) (Werner, 2003).

Activated leukocyte compositions (ALC) for repairing or promoting the prevention and healing of wounds, and methods of making and using ALCs, are described in U.S. Patent No. 8,574,902 and related U.S. Patent Application Publication Nos. 2013/0071465 and 2014/0004091 , each of which is incorporated by reference in its entirety. As these references describe, after production the ALC is typically stored at room temperature, for example, at approximately 20-24° C. This temperature is maintained throughout the ALC production procedure (except for incubation steps). The ALC is also maintained at this temperature during any periods of shipping, up until and including the point in time that the ALC is administered to a subject with a wound in need of treatment. Maintained at room temperature, the ALC has a commercially useful shelf life of up to 50 hours measured from the time of blood donation, or 27 hours after completing production of the ALC. This relatively short shelf life places a logistical constraint on the manufacturing, shipping and distribution, and therapeutic use of the ALC. Such problems are particularly acute in the case of large-scale ALC production required to address market demands, which implies a large number of participating donors and relatively long-term storage before the product is administered to patients. A suitable storage solution is also critical since the ultimate therapeutic efficacy for the ALC depends on the activity profile of the leukocytes, which may change over time as the various subsets of cells in the ALC respond to storage conditions in different ways. Therefore, it is desirable to provide a storage solution that allows the ALC to be stored for extended periods of time before its therapeutic use and, at the same time, provides a good retention of leukocyte stability and activity over time.

BRIEF SUMMARY OF THE DISCLOSURE

The present embodiments described are on the discovery that storing an activated leukocyte composition (ALC) under at temperatures below room temperature provides for good retention of leukocyte stability and activity of the ALC for extended periods of time.

There is disclosed a method for the storing an activated leukocyte composition comprising providing the activated leukocyte composition, and maintaining the activated leukocyte composition at a temperature ranging from above 0° C to below about 10° C. In some of the disclosed methods, the temperature ranges from about 2° C to about 8° C. In other disclosed methods the temperature ranges from about 4° C to about 6° C. In one method, the temperature is about 5° C.

In some of the disclosed methods, the activated leukocyte composition is maintained at a temperature ranging from 2° C to about 8° C for about 1 , 2, 3, 4, 5, 6, or 7 days. In other disclosed methods, the activated leukocyte composition is maintained at a temperature ranging from about 4° C to about 6° C for about 1 , 2, 3, 4, 5, 6, or 7 days. On other disclosed methods, the activated leukocyte composition is maintained at a temperature of about 5° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

Also disclosed herein is an activated leukocyte composition stored at a temperature ranging from above 0° C to below about 10° C. In some embodiments, the composition comprises serum. In other embodiments, at least 85% of cells in the activated leukocyte composition remain viable after seven days of storage. In other embodiments, at least 90% of cells in the activated leukocyte composition remain viable after seven days of storage.

Also disclosed is a method for prolonging the activity of leukocytes in an activated leukocyte composition comprising providing the activated leukocyte composition, and

maintaining the activated leukocyte composition at a temperature ranging from above 0° C to below about 10° C. In some embodiments, the temperature ranges from about 2° C to about 8° C. In other embodiments, the temperature ranges from about 4° C to about 6° C. In another embodiment, wherein the temperature is about5° C.

in some of the disclosed methods, the activated leukocyte composition is maintained at a temperature ranging from 2° C to about 8° C for about 1 , 2, 3, 4, 5, 6, or 7 days. In other disclosed embodiments, the activated leukocyte composition is maintained at a temperature ranging from 4° C to about 6° C for about 1 , 2, 3. 4, 5, 6, or 7 days. In additional disclosed methods, the activated leukocyte composition is maintained at a temperature of about 5° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

Also disclosed are activated leukocyte compositions comprising leukocytes having prolonged activity made according to the methods disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the leukocyte (or white blood cell, hereinafter WBC) viability of three final product (FP) batches before and after storage at 2-8° C. WBCs viability was measured at time "0" (i.e., before storage) and after storage and plotted against post-production days. Linear regression analysis for WBC viability was performed. The dotted lines represent the 95% confidence limits of the linear regression analysis for the plotted data line for each FP batch.

FIG. 2 illustrates the leukocyte (WBC) concentration of three FP batches before and after storage at 2-8° C. WBCs concentration was measured with a Cell Dyn Analyzer at time "0" (i.e., before storage) and after storage and plotted against post-production days. Linear regression analysis for WBCs concentration was performed. The dotted lines represent the 95% confidence limits of the linear regression analysis for the plotted data line for each FP batch.

FIG. 3 illustrates the lymphocyte percentage of the three FP batches before and after storage at 2- 8° C. Lymphocyte percentage was measured with a Cell Dyn Analyzer at time "0" (i.e., before storage) and after storage and plotted against post-production days. Linear regression analysis for lymphocyte percentage was performed. The dotted lines represent the 95% confidence limits of the linear regression analysis for the plotted data line for each FP batch.

FIG. 4 illustrates the granulocyte activation (potency) of the three FP batches before and after storage at 2-8° C. Leukocytes were stained with the antibody against activated form of CD 1 l b (clone CBRMl/15) conjugated to phycoerythrin (PE) at time "0" (i.e., before storage) and after storage. Mean Florescence Intensity (MFI) of the CD 1 lb staining was plotted against post-production days. Linear regression analysis for MFI was performed. The dotted lines represent the 95% confidence limits of the linear regression analysis for the plotted data line for each FP batch.

FIG 5 provides an analysis of CD1 l b expression of ALCs before and after storage.

FIG. 6 provides an analysis of IL-8 content of ALCs before and after storage.

FIG. 7 provides an analysis of CD62L expression of ALCs before and after storage.

FIG. 8 provides an analysis of CD68 expression of ALCs before and after storage.

FIG. 9 provides an analysis of HLA-DR expression of ALCs before and after storage.

FIG. 10 shows the effect on WBC concentration when activated leukocytes are stored at room temperature. FIG. 1 1 shows effect on leukocyte percentage when activated leikocytes are stored at room temperature.

DETAILED DESCRIPTION

Blood is defined herein as whole blood or any of its constituent parts (e.g., plasma, leukocytes, platelets or red blood cells). The amounts of platelets and red blood cells that may be present in the ALC may be lower than that in whole blood.

The term "about" as used herein in connection with any and all values (including lower and upper ends of numerical ranges) means ±10%.

The starting materials for producing the ALCs may be obtained from several sources. Whole blood or one or more components thereof (e.g., leukocytes and plasma) may be obtained from autologous or allogeneic sources. In one embodiment, the blood sample is collected from the patient who will ultimately be treated with the ALC, which is referred to herein as an autologous blood sample or source. In embodiments wherein the source(s) i.e., the blood or its components, is obtained from an individual other than the intended ALC recipient, which is referred to as an allogeneic blood sample or source, these starting materials may be conveniently obtained from a blood bank. The samples may be screened by the blood bank for blood type (ABO, Rh), irregular antibodies to red cell antigens, and transfusion- transmittable diseases. More specifically, screening can be conducted with antibodies using an Abbott PRISM instrument against: Hepatitis B, C, HIV 1/2, HTLV and Syphilis (-HCV; HbsAg; anti-HIV 1/2 0+; and anti-HTLV I/II). The samples can also be screened for HIV, HCV and HBV by molecular methods (NAT-Nucleic Acid Testing). Molecular screening can be accomplished using commercially available instrumentation, e.g., the TIGRIS system of Chiron.

In these embodiments involving allogeneic sources, the samples can be obtained from donors with the same blood type as the intended ALC recipient. Alternatively and as further described herein, plasma samples can be obtained from donors with AB+ blood and the leukocytes can be obtained from patients with O— blood. Patients with AB+ blood are universal donors for plasma and patients with O- blood are universal donors for leukocytes. In still other embodiments, the leukocytes and/or plasma may be of any blood type. The plasma used can be fresh, stored (e.g., at 1 -6°C. for less than 24 hours), dried, or otherwise pre-treated (e.g., pathogen-reduced plasma and solvent/detergent (SD) treated plasma). The plasma can be fresh or stored at 1 -6° C. for less than 24 hours, or Fresh Frozen Plasma, or Dried Plasma, or Pathogen-Reduced Plasma, or Solvent/Detergent (SD) Treated Plasma. In addition to allogeneic sources, the cells may be obtained from autologous samples, by apheresis, or from umbilical cord blood. Regardless of the source, all necessary processing of the sample(s) can be carried out without the need for highly specialized equipment.

One method of making the ALCs is described in U.S. Patent No. 8,574,902, which is incorporated by reference in its entirety.

In some embodiments, the method for making an ALC includes the steps of subjecting leukocytes, which may be obtained from a sample of whole human blood, to a first incubation for a period of time and at a temperature which allows the leukocytes to become activated, which in some embodiments, is about 8 to about 20 hours, and at room temperature. After incubation, the leukocytes are contacted with a physiologically acceptable aqueous solution such as sterile, distilled water, to initiate hypo-osmotic shock, followed by contacting the shocked leukocytes with a physiologically acceptable salt solution to restore isotonicity. This ALC may be used therapeutically. However, in some embodiments, separate and substantially concurrent with the first incubation of the leukocytes, a sample of plasma, which may be obtained from the same or different whole blood sample (i.e., from the same or a different human), is contacted with a coagulating agent at about 37° C concurrent with the leukocyte incubation, which in some embodiments, is about 8 to about 20 hours, followed by separating serum from the coagulated plasma sample. The leukocytes are resuspended in serum collected from the coagulated plasma sample, thus forming the ALC. After the first incubation, the leukocytes may be further subjected to a second incubation for about 60 to about 120 minutes at about 37° C.

The ALC will include leukocytes, e.g., granulocytes, monocytes and lymphocytes. Granulocytes include neutrophils, eosinophils and basophils. In its broadest sense, the leukocyte population of the ALC generally contains about 40% to about 90% granulocytes, about 5% to about 20% monocytes and about 5% to about 30% lymphocytes. Specific amounts of the cells may differ based on the analysis techniques employed. When analysis is performed using Fluorescence-Activated Cell Sorting (FACS) (e.g., using a side-scatter versus a forward-scatter dot plot analysis), the leukocyte composition generally contains about 55% to about 80% granulocytes; about 5% to about 15% monocytes and about 5% to about 30% lymphocytes, and in some embodiments, comprises about 58-76% granulocytes; about 5-1 1 % monocytes and about 9-23% lymphocytes. When analysis is performed using a Cell Dyn Analyzer, the leukocyte composition generally contains about 50% to about 90% granulocytes; about 5% to about 15% monocytes; and about 10% to about 25% lymphocytes. The subpopulation of lymphocytes in the ALC may confirm the following cells in the general ranges as follows: about 7% to about 25% B cells (CD19+); about 20% to about 30% NK cells (CD3-/CD56+), about 40% to about 60% T cells (CD3+); about 0.1 % to about 30% of NKT cells CD3+/CD56+, about 8% to about 20% of T helper cells (CD4+/CD3+), and about 20% to about 30% of CD8+/CD3+ cells. In some embodiments, the lymphocyte subpopulation is enriched with at least 9% CD56+ cells (CD3-/CD56+; CD3+/CD56+; CD3+/CD56+/CD8+), the amount of the T helper lymphocytes (CD4+/CD3+) is decreased to less than 20%, and/or the ratio of T-helper to T-suppressor cells (CD4+/CD3+: CD8+/CD3+) is less than 0.8. The ALC may also include stem and progenitor cell populations. For example, the ALC may further contain mesenchymal stem cells and endothelial progenitor cells, each in amounts ranging from about 0.1 % to about 5.0% of the total cell population in the ALC.

For purposes of this disclosure, leukocyte activation is defined as a process involving at least one stage, by which the cells (leukocytes) undergo a transition from a quiescent to a functionally active state which is accompanied by synthesis of biologically active substances or translocation of pre-synthesized substances, e.g., cytokines including IL-8, from the cytoplasm to the cellular membrane or their release into extracellular medium (which in this case is serum). Activation of leukocytes in vivo may involve migration of the cells closer to and along the blood vessel wall, which is mediated by P-selectin (and increased CD42b expression), increased adhesion of leukocytes to the endothelial wall, spreading and extravasation, which is mediated to a large degree by activated CD 1 l b that interacts with endothelial ligands ICAM-1 and ICAM-2; migration to the focus of inflammation via interaction with extracellular matrix proteins, e.g., laminin) and functional responses to inflammatory stimuli such as respiratory burst, degranulation, phagocytosis and release of cytokines. Activation of the leukocytes may also be indicated by increased expression of activated form of CD 1 l b receptor on leukocyte populations including granulocytes, monocytes and lymphocytes. CD62L is an adhesion receptor from a selectin family. It is constitutively expressed on all classes of leukocytes including granulocytes, monocytes and lymphocytes. Upon activation, leukocytes rapidly shed off CD62L from their surface. CD62L is a plasma membrane protein which is shed during activation and thus decreases with cell activation. In some embodiments, activated leukocytes may exhibit decreased levels of CD62L. In some embodiments, activated leukocytes may also exhibit increased levels of CD68, a lymphocyte-specific marker, increased levels of cell surface receptor HLA-DR, and/or increased production of IL-8. Yet other indicia of leukocyte activation may include increased production of one or more of proteins or polypeptides, lipids, sugars, oxygen radicals and other biochemical moieties that function as adhesion molecules, cytokines in addition to IL-8, growth factors, enzymes, transcription factors and cell signaling receptors and mediators. Altered expression levels of any of these molecules is assessed from the standpoint of the leukocytes contained in a "fresh buffy coat" (as described herein), without being subjected to an incubation.

It is a purpose of this disclosure to provide storage solutions that allow activated leukocyte compositions (ALCs) to be stored for an extended period of time while retaining leukocyte stability and activity over time.

Retaining leukocyte activity is defined herein as the leukocytes remaining activated once they are activated. In some embodiments, leukocyte activity is indicated by the expression level of cell surface markers of activation. In some embodiments, the indicators of retained leukocyte activity, based the expression level cell surface markers of activation, include but are not limited to greater expression levels of GD I lb, greater expression levels of CD68, greater expression levels of HLA-DR, and lower expression levels of CD62L compared to those of the leukocytes contained in a "fresh buffy coat" (as described herein), without being subjected to an incubation.

In some embodiments, leukocyte activity is also indicated by secretion of certain cytokine(s), growth factor(s), or soluble mediator(s). In other embodiments, an ALC retaining leukocyte activity has a concentration of at least one cytokine(s), growth factor(s), or soluble mediator chosen, for example, from IL-8, PDGF, Ang- 1 , VEGF, IL-6, TNF-alpha, or IL-Ιβ, that is at least about 1.25, 1 .5, 2, 3, 4, 5, 6, 7, 8. 9, 10 or more times the concentration of that same cytokine, growth factor, or soluble mediator in the same medium used to prepare the incubation composition that resulted in that ALC, but prior to contact of the medium with an activated leukocyte composition.

Retaining leukocyte stability is defined herein as the ALC meeting the stability specifications as described herein. Stability specifications of the ALC include at least high viability of leukocytes, high concentration of leukocytes, high percentage of lymphocytes, and high percentage of activated granulocytes.

In some embodiments, the ALCs, stored according to the present disclosure, may contain at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or greater, viable leukocytes based on this total number of leukocyte cells in the ALC (including all sub-ranges thereof).

In some embodiments, the ALCs, stored according to the present disclosure, may have a leukocyte concentration (cells/ml) of at least l xl O⁶, 1.5xl 0⁶, 2x l 0⁶, 2.5x 10⁶, 3x l 0⁶, 3.5xl 0⁶, 4x l 0⁶, or greater (including all sub-ranges thereof).

In some embodiments, the ALCs, stored according to the present disclosure, may contain at least

14%, 16%, 18%, 20%, 22%, 24%, 26%, 28%, 30%, 32%, 34%, 36%, 38%, 40% or greater, lymphocytes based on this total number of leukocyte cells in the ALC (including all sub-ranges thereof).

50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%. 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, or higher, CD 1 lb(+) granulocytes, relative to the total granulocyte population in the ALC (including al l sub-ranges thereof).

In some embodiments, the activated leukocyte composition (ALC) is placed and stored under cold temperature immediately after production.

In some embodiments, the activated leukocyte composition (ALC) is placed and stored under cold temperature after being stored at room temperature or other suitable temperature.

For purposes of this disclosure, "cold temperature" refers to a temperature that is below room temperature, for example, above 0° C and below about 10° C. In some embodiments, the range is from about 2° C to about 8° C, and in other embodiments from about 4° C to about 6° C. In one embodiment, the temperature is 5° C.

For purposes of the present disclosure, "room temperature" refers to a temperature in the range of about 18° C to about 28° C, and more typically, about 20° C to about 24° C.

In one embodiment, the activated leukocyte composition (ALC) is placed and stored under cold temperature, wherein the cold temperature is provided and maintained by a refrigerator or any suitable temperature controlling device.

In one embodiment, the ALC, stored according to the present disclosure, has a shelf life (i.e., can be stored prior to use) extending up to 8 days following end of production, for example about 1 , 2, 3, 4, 5, 6, 7 or more days following its production.

In one embodiment, the ALC, stored according to the present disclosure, has a shelf life extending up to 10 days following collection of the blood sample or blood samples used to prepare the ALC, that is, about 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more days following collection of the blood sample or blood samples used to prepare the ALC. The ALC, stored according to the present disclosure, can be used and applied according to the disclosure in U.S. Patent No. 8,574,902.

For example, the ALC is useful in promoting healing in a multitude of wound types. Although in practice it may be used in combination with other treatment modalities, it does not require them to achieve effective wound healing. The ease of application, e.g., with a standard syringe or similar application device, makes the ALC compositions safe and easy to use.

Wounds amenable to treatment with the ALC are typically in the form of burns, punctures, and cuts or tears of the living tissues. Wounds of the skin can penetrate the epidermis, dermis or in the case of full-thickness wounds, the subcutaneous tissue. Thus, representative types of wounds amenable to treatment with the compositions and methods of the present disclosure include burns (e.g., caused by exposure to fire or an agent that is highly caustic to skin such as radiation), ulcers (e.g., decubital or pressure ulcers; venous ulcers and diabetic ulcers), deep sternal wounds, e.g., following open heart surgery; and post-operative wounds following abdominal, orthopedic and any other types of surgery. Other wounds are those which result from trauma such as incurred during combat or other violent activity, including wounds caused by gun shots, knives, or any other object able to cause a cut or tear in the skin. Wounds of the oral cavity (e.g., teeth), as well as wounds that arise as a side-effect of medication or as a symptom of various pathologies (e.g., sores associated with Kaposi's Sarcoma), as well as internal wounds (e.g., ruptures of muscle tissue such as anal fissures), and wounds or lesions to the gastrointestinal tract, such as ulcers in the stomach or intestines) may also be amenable to treatment with the present disclosure. In addition to providing a wound-healing effect, the ALC may also provide a benefit of inhibiting onset of infection following such therapy, particularly in the context of surgery.

The ALC may also be used to treat any wounds exacerbated by vascular insufficiency. Vascular insufficiency, for purposes of this disclosure, refers to inadequate blood circulation resulting in insufficient perfusion to the afflicted areas. Such insufficiency can be caused by trauma (e.g., damage to the vasculature adjacent to a skeletal fracture), or various pathologies (e.g., diabetes and atherosclerosis). In either instance, whether trauma or disease induced, vascular insufficiency decreases the likelihood of effective wound healing. The ALC may be useful in improving wound healing outcomes in these patients and should be administered according to the methods described herein. Additionally, treatment algorithms should not be limited by the severity or type of wound, or the extent of vascular insufficiency. ALC may be more efficacious in patients presenting with the most severe wounds and vascular insufficiency.

In general, application of the activated leukocyte composition is accomplished by means of one or more injections of the ALC at a single or multiple sites, using a suitable syringe (e.g., a 2 ml syringe fitted with an 1 8 G or 25 G needle) directly into the wound or the tissue surrounding the wound. In some embodiments, injection occurs about every one centimeter to about every three centimeters for the entire length of the wound. At each injection site, about 0.1 to about 0.3 ml of ALC is injected. ALC compositions of the present disclosure contain leukocytes in a concentration that generally ranges from about 2 10⁶ cells/ml to about 4 l0⁶ cells/ml. For injection into the wound, one may use a Luer-Lock syringe or any other commercially available syringe that has a locking mechanism between the syringe and the needle. The biological space of a wound, particularly a pressure wound, is often limited. When injecting into a wound, there is a risk of pressure causing the syringe to separate from the needle. Using a locking syringe eliminates this risk.

When injection into the wound tissue is not possible, the ALC can be applied directly into the cavity of the wound. Application in this method can be done using direct application with a syringe or tubing.

The ALC may be applied to or around the wound site with the aid of a dressing. Dry dressings include gauze and bandages, non-adhesive meshes, membranes and foils, foams, and tissue adhesives. Moisture-keeping barrier dressings include pastes, creams and ointments, nonpermeable or semipermeable membranes or foils, hydrocolloids, hydrogels, and combination products. Bioactive dressings include antimicrobial dressings, interactive dressings, single-component biologic dressings, and combination products (e.g., ointments, gels, fibrin sealant, growth and angiogenic factors (e.g., PDGF, BEGF, collagen)). In some embodiments, the wound is packed with sterile gauze soaked in the ALC. The dressing, such as sterile gauze pads, may be saturated with compositions such as Lactated Ringer (Hartman) Solution, alginate containing dressing, polyurethane dressing or carboxymethylcellulose dressing, which is applied to cover the wound, followed by application of dry dressing. If the subject wound is highly infected, then silver dressings such as Silverlon can be applied. The choice of post- injection dressing is based on the determination of the clinician. Commercial availability, history of past clinical success, and patient tolerance are all factors to be considered in the selection of a wound dressing. The dressing may be removed periodically, e.g., typically after about 24 hours, in order to irrigate the wound e.g., with sterile water and soap.

In another embodiment, the ALC composition is combined with a physiologically inert and/or resorbable matrix or scaffold prior to administration. The matrix or scaffold may be formed from any material suitable for implantation into a person. For example, the matrix or scaffold may comprise any biocompatible material including collagen, hyaluronic acid, or gelatin, or combinations thereof. The collagen may be obtained from any source, including collagen prepared from human tissue or the tissue of other collagen-producing mammals. The combination of the ALC composition and matrix/scaffold material may form a gel or putty that is administered to a person by means of a press fit, or by injection. This allows for a sustained delivery of the ALC into the site which benefits the patient in that the cells have a longer period in situ. In some embodiments, the mixture of the ALC composition and matrix/scaffold material is prepared commercially and provided to a health-care provider pre-mixed. In other embodiments, the health care provider mixes the ALC composition and the matric/scaffold prior to administration to a person.

The ALC compositions may be applied to the wound once or more than once, e.g., after 4 weeks, once a clinician determines whether another application is necessary. Factors that may be taken into account include increased wound dimensions (width, length and depth), suppuration, pyrexia or any other sign or symptom indicating a recalcitrant infection such that re-treatment is warranted. In addition to re- treatment, referral for surgical debridement may be indicated at any point the clinician deems appropriate.

The ALC may be used in conjunction with any other conventional wound treatment, such as negative pressure, warming (therapeutic heat), electrical stimulation, magnetism, laser phototherapy, cycloidal vibration therapy and ultrasound. It also can be used with biological therapy such as larva therapy, skin substitutes, cultured keratinocytes (Epicel, Genzyme biosurgery), human dermal replacement (Dermagraft, Organogenesis Inc. Inc.), cadaver derived processed dermis ( Alloderm, Life Cell Corporation), Bilayered Skin Equivalent (Apligraf, Organogenesis Inc.), TransCyte (Smith and Nephew Inc.), Growth Factors (PDGF is currently the only growth factor licensed for topical use), and fibrin sealant. In some embodiments, the ALC is used in conjunction with negative pressure wound therapy (NPWT) (one example being the V.A.C., which is a commercially available wound therapy manufactured by KC1). Negative pressure therapy promotes wound healing by applying negative pressure to a wound. In these embodiments, ALC may be applied to a wound prior to negative pressure therapy. In yet other embodiments, the ALC is used in conjunction with hyperbaric oxygen therapy (Thackham, 2008) or ozone therapy. For example, the ALC can be applied to a wound just prior to a patient receiving hyperbaric therapy. The ALC may also be used in conjunction with low-energy shock wave therapy (e.g., impulses of about 0.1 mJ/mm²; 5 Hz) per centimeter of wound length). See, e.g., Dumfarth, et al., Ann. Thorac. Surg. 86: 1909-13 (2008).

After treatment, the wounds may be evaluated for length, width and height measurements.

Typically, a wound is considered healed when all measurements of these parameters are negligible. The ALC may also provide an analgesic effect.

The ALC is particularly useful in wounds including diabetic foot ulcers and decubital ulcers. Decubital ulcers are pressure ulcers caused by impeded blood flow, usually due to prolonged pressure on a particular area. (Berlowitz, 2007) Decubital ulcers cause morbidity and mortality in elderly people. At least 48% of stage 1 V pressure ulcers remain unhealed after one year of treatment. (Girouard, 2008). Patients suffering from decubiti also commonly have co-morbid pathologies such as diabetes and hypertension. These pathologies further complicate the successful treatment of decubiti.

In one embodiment for treating decubital ulcers, the composition is aspirated into a sterile syringe of any size, using an 18-gauge (18G) needle. Aspiration is performed slowly to minimize damage to the cells. While the size of the syringe and needle are by no means limiting, a large gauge needle is useful for aspiration. This facilitates the transfer and reduces cell damage.

Application of the ALC to the ulcer comprises injecting the composition into the wound. The entire sample in the syringe can be deployed and the clinician can choose to administer additional ALC if it is determined to be necessary based on clinical parameters.

The 18G needle used for aspiration is exchanged with a needle ranging in size from 22-35 G.

The ALC may be injected into the wound in various locations. In one embodiment, injection occurs about every one centimeter to about every three centimeters for the entire length of the wound. At each injection site, 0.1 -0.3 ml of ALC is injected. In another embodiment, the entire syringe can be injected at one time into a single site within the wound.

Aspect(s) of the present disclosure will now be described in accordance with the following non- limiting examples.

Example 1 : Production of Final Product Batches

Three ALC final product (FP) batches (referred to herein as Batches 1 , 2, and 3) from three different donors were prepared and stored in storage bags as follows.

Leukocytes and plasma from a typical blood component separation were obtained from a blood bank. The leukocytes and plasma were transferred into a custom bag set, such as described in U.S. Patent 8,574,902.

Buffered CaCl₂ solution was added to bag containing the donor plasma. The plasma bag was then placed in an incubator at 37 ± 1 °C for 12±2 hours. During this incubation the plasma converted into serum. In parallel, the leukocytes are incubated at 20-24°C for the same 12±2 hour period.

Following this incubation, the bag set was removed from the incubator and pharmaceutical grade sterile water (200 mL) was added to the leukocytes and incubated for 45±5 seconds followed by the addition of buffered 9% NaCl solution to the leukocyte suspension to re-establish the isotonicity of the leukocyte preparation. The bags were than centrifuged at 1820 X g, at 20-24°C for 5 minutes to pellet the leukocytes and to enable subsequent removal of the buffered sodium chloride supernatant from the leukocyte bag. After centrifugation, the supernatant from the leukocyte-containing bag was transferred into a waste bag.

Following removal of the supernatant, the leukocyte cell pellet was suspended in the autologous serum and the cell suspension was then incubated for 90±5 minutes at 37± 1 °C. After this incubation, the bag set was removed from the incubator and the serum was removed from leukocytes and fresh serum was added to the leukocytes.

A sample of the leukocyte suspension was removed to permit a cell count. The leukocyte concentration was adjusted by adding a portion of serum so that the final cell concentration was between 3.6 x 10⁶ to 4 x 10⁶ cells/mL.

All three FP batches were stored immediately after production at 2-8° C. Testing of the stored FP batches was performed within 20 minutes after they were pulled out the cold storage. During the 20- minute period, the storage bags containing the FP were gently massaged by hand, gently resuspended using a sterile syringe with 21 G needle, and sampled. The three FP batches were sampled immediately after production and before storage (time "0") and at 1 , 3, 4, 5, 6 and 7 days post-production. These batches were tested for leukocyte viability (Example 2), leukocyte concentration (Example 3), lymphocyte percentage (Example 4), and granulocyte activation (Example 5).

Example 2: Analysis of Leukocyte Viability

The leukocyte (WBC) viability of the three FP batches before and after storage at 2-8° C was tested. Control cells were incubated at room temperature (20°-24° C) for 27 hours. These conditions were chosen based on prior knowledge that ALCs incubated at room temperature for more than 27 hours are no longer suitable for commercial use. The number of viable WBCs contained within the total WBCs population, expressed as a percentage, was measured. The results are presented in FIG. 1 A.

Five quantitative attributes were plotted using linear regression analysis with 95% confidence limit. Only NC-I-032 demonstrates significant linear degradation (r2=0.73) with the lower 95% confidence limit intersecting 85% viability after 10.2 days storage at 2-8° C. Batches 2 and 3 demonstrated a slight non-significant decrease (r2=0.12 and 0.06, respectively) with the lower 95% confidence limit intercepting with the lower acceptance criterion after 37.2 and 15.5 days at 2-8° C, respectively.

FIG I B shows an analysis of white blood cell viability before and after storage. Batch 1 demonstrated slight non-significant decrease (r2=0.1 1 ) with the lower 95% confidence limit intercepting with the lower acceptance criterion after 57.8 hours at RT. Batches 2 and 3 demonstrated slight nonsignificant increase (r2=0.87 and 0.06 respectively). Note that separate viability counts, and not the final reported results, were used for the statistical analysis.

Example 3: Analysis of Leukocyte Concentration

The three FP batches were tested for leukocyte (WBC) concentration before and after storage at

2-8°C with a Cell Dyn Analyzer. The results are presented in FIG. 2. Based on the statistical analysis, the shortest stability is determined by Batch 1. The lower 95% confidence limit of Batch 1 intersects with the lower cutoff value (2.0 10^b cells/ml) after 7.7 days storage at 2-8° C. The lower 95%> confidence limits of Batches 2 and 3 intercept with the lower cutoff value after 1 1.2 and 10.8 days at 2-8° C, respectively. All three batches tested in this study demonstrated significant linear degradation (r²=0.88, 0.87, and 0.88, respectively).

Example 4: Analysis of Lymphocyte Percentage

The lymphocyte percentage of the three FP batches before and after storage at 2-8° C was tested. The number of lymphocytes contained within the total leukocytes population, expressed as a percentage, was measured with a Cell Dyn Analyzer. The results are presented in FIG. 3.

Only Batch 3 demonstrated significant linear regression (r2=0.70), which shows a decrease in the lymphocyte percentage over time with no interception with the upper cutoff criterion of 46%. Batches 1 and 2 demonstrated a slight non-significant (r2=0.12 and 0.10 respectively) increase with the upper 95% confidence limit intercepting with the upper cutoff criterion after 34.7 and 29.2 days at 2-8° C, respectively.

Example 5: Analysis of Granulocyte Activation

Granulocyte activation, which serves as a measurement of potency, of the three FP batches was tested using CD 1 1 b as an activation marker. Leukocytes were stained with an antibody against an activated form of CD1 lb (clone CBRM l/15) conjugated to phycoerythrin (PE) before and after storage at 2-8° C. Mean Florescence Intensity (MFI) of the anti-CDl l b staining was plotted against post production (storage) time. Linear regression analysis for MFI including 95% confidence interval for the individual predicted value was performed. The results are presented in FIG. 4. All three batches tested in this study demonstrate non-significant linear degradation (r²=0.48, 0.01 and 0.01 , respectively). Based on the statistical analysis the shortest stability is determined by Batch 1 . The lower 95% confidence limit of Batch 1 intersects with the lower cutoff criterion (50 MFl) after 12.0 days storage at 2-8° C. The lower 95% confidence limit of Batch 2 intercepts with the lower cutoff criterion after 18.5 days at 2-8° C. The lower 95% confidence limit of Batch 3 never intercepts with the lower cutoff criterion.

Example 6: Comparative Analysis of ALCs Stored at Different Temperatures

Three FP batches from three different donors were prepared and stored in storage bags as described in Example 1. The activation status of the cells of the starting material (Fresh Buffy Coat, or FBC) and those of the FP were tested before storage (FP), 27 hours post-production at room temperature (RT; 20°-24° C), and 27 hours, 1 , 3, 4, 5, 6, and 7 days post production at 2-8° C (RF).

The activation status was measured by analyzing cell surface markers of activation (CD1 lb, CD62, CD68 and HLA-DR), and by measuring pro-inflammatory cytokine secretion. For example, IL-8 secretion (pg/ml) into Roswell Park Memorial Institute medium (RPMI) containing serum, into serum, and internal IL-8 (i.e., not secreted), was measured. Cell surface markers of activation (CD1 l b, CD62L, CD68, and HLA-DR) were stained with corresponding antibodies and their expression levels were analyzed. The viability and sterility of the cells was also tested to show that potency observed reflects the activity of live cells, and that cell activation was due to the hypoosmotic-shock step rather than contamination. The results are presented in Tables 1 -3.

The analysis of the three FB batches revealed that the presently disclosed methods achieve at least the unexpected results of stable and sustained production and release of IL-8 when stored at 2-8° C for 7 days post-production.

TABLE 1

Activation Analysis of Batch 4

Time

FBC FP 27RT 27RF 3D 4D 5D 6D 7D point

Viability NA 99 96 94 94 93 92 96 90

CDl lb 32 271 249 262 354 276 283 296 181

CD62L 409 41 18 21 8 5 4 J

CD68 4 14 12 1 1 1 1 10 12 13 13

HLA-DR 98 243 484 166 161 178 173 173 172

IL-8

36 732 2584 987 1242 1297 1603 1308 NA RPMI

IL-8

1 1 1 1647 4443 2510 1752 2457 2843 2433 4050 Serum

IL-8

9 4685 14698 3849 4730 4589 61 16 7443 6757 Content TABLE 2

Activation Analysis of Batch 5

Time

FBC FP 27RT 27RF 3D 4D 5D 6D 7D point

Viability NA 98 98 98 97 95 99 90 90

CDl lb 29 316 366 300 254 292 250 245 243

CD62L 348 38 j 20 6 4 2 2 1

CD68 6 1 1 9 1 1 1 1 10 10 9 10

HLA-DR 78 205 332 154 145 165 193 165 157

IL-8

58 932 4504 1352 1262 1400 1061 1059 1057 RPMI

IL-8

99 2725 7466 2942 4534 391 1 2804 2294 Serum

IL-8

5 3761 29700 4589 6028 6249 5705 5376 5641 Content

TABLE 3

Activation Analysis of Batch 6

Time

FBC FP 27RT 27RF 3D 4D 5D 6D 7D point

Viability NA 99 99 98 96 94 93 94 86

CDl lb 5 263 242 281 273 251 288 272 182

CD62L 593 95 31 48 34 9 10 1 1 2

CD68 4 14 13 1 1 1 1 14 14 15 15

HLA-DR 98 227 376 151 136 156 195 156 202

IL-8

76 552 4048 789 725 791 901 928 975 RPMI

IL-8

1 13 2859 10328 3402 2236 2252 2550 2097 1951 Serum

IL-8

j 1695 15228 2145 2349 3312 2564 2752 3261 Content Mean and standard deviations for the three batches are shown in the following tables:

As shown in FIG. 5, the fluorescent intensity of all three batches (i.e., Batches 4, 5, and 6) stained with anti-CD l l b antibody were at least 30% higher at all time points as compared to the intensity of the Fresh Buffy Coat (FBC) stained with anti-CDl l b.

As shown in FIG. 6, the IL-8 concentrations for these three batches were at least 30% higher at all time points as compared to the IL-8 concentration in the Fresh Buffy Coat (FBC).

As shown in FIG. 7, the fluorescent intensity of all three batches stained with ant i-CD62L antibody were at least 30% lower at all time points as compared to the intensity of the Fresh Buffy Coat (FBC) stained with anti-CD62L.

As shown in FIG. 8, the fluorescent intensity of all three batches stained with anti-CD68 antibody were at least 30% higher at all time points as compared to the intensity of the Fresh Buf As shown in FIG. 9, the fluorescent intensity of all three batches stained with anti-HLA-DR antibody were at least 30% higher at all time points as compared to the intensity of the Fresh Buffy Coat (FBC) stained with anti-HLA-DR antibody.

These analyses reveal that storage of the ALCs at cold temperatures maintained the viability of the cells, and the potency of the ALC for a longer period compared to ALCs stored at room temperature.

Example 7: Analysis of Activated Leukocytes Stored at Room Temperature

Activated leukocytes made according to the methods disclosed herein were tested following activation (T = 0), and after 1 , 3, and 7 days storage at room temperature. The following data were obtained:

As shown in FIG. 10, significant degradation was observed in WBC concentration over the storage period of time. The degradation line exceeded 2.0xl 0⁶ cells/ml after 2 days storage at RT.

As shown in FIG. 1 1 , a significant increase was observed in lymphocyte percentage over the storage period of time. The percentage exceeded 43% after 2 days storage at RT.

Claims

What is claimed is:

1. A method for the storing an activated leukocyte composition comprising:

providing the activated leukocyte composition; and

maintaining the activated leukocyte composition at a temperature ranging from above 0° C to below about 10° C.

2. The method of claim 1 , wherein the temperature ranges from about 2° C to about 8° C.

3. The method of claim 2, wherein the temperature ranges from about 4° C to about 6° C.

4. The method of claim 3, wherein the temperature is about 5° C.

5. The method of claim 2, wherein the activated leukocyte composition is maintained at a temperature ranging from 2° C to about 8° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

6. The method of claim 3, wherein the activated leukocyte composition is maintained at a temperature ranging from about 4° C to about 6° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

7. The method of claim 4, wherein the activated leukocyte composition is maintained at a temperature of about 5° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

8. An activated leukocyte composition stored according to the method of claim 1.

9. The activated leukocyte composition of claim 8, wherein the composition comprises serum.

10. The activated leukocyte composition of claim 8, wherein at least 85% of cells in the activated leukocyte composition remain viable after seven days of storage.

1 1. The activated leukocyte composition of claim 10, wherein at least 90% of cells in the activated leukocyte composition remain viable after seven days of storage.

12. A method for prolonging the activity of leukocytes in an activated leukocyte composition comprising:

providing the activated leukocyte composition; and

13. The method of claim 12, wherein the temperature ranges from about 2° C to about 8° C.

14. The method of claim 13, wherein the temperature ranges from about 4° C to about 6° C.

15. The method of claim 14, wherein the temperature is about 5° C.

16. The method of claim 12, wherein the activated leukocyte composition is maintained at a temperature ranging from 2° C to about 8° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

17. The method of claim 16, wherein the activated leukocyte composition is maintained at a temperature ranging from 4° C to about 6° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

18. The method of claim 17, wherein the activated leukocyte composition is maintained at a temperature of about 5° C for about 1 , 2, 3, 4, 5, 6, or 7 days.

19. An activated leukocyte composition comprising leukocytes having prolonged activity made according to the method of claim 12.