Patent Publication Number: US-9833475-B2

Title: Enhanced anabolic cytokine production and delivery system

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 14/188,899, filed Feb. 25, 2014, which claims the benefit of U.S. Provisional Application No. 61/771,321, filed Mar. 1, 2013, the disclosure of which is incorporated by reference in its entirety herein. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to methods and apparatus for producing therapeutically active proteins with decreased catabolic cytokines. 
     BACKGROUND OF THE INVENTION 
     The pathogenesis of osteoarthritis involves multiple etiologies, including mechanical, biochemical, and genetic factors that contribute to the imbalance in the synthesis and destruction of articular cartilage. Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta or IL-1β) and tumor necrosis factor-a are the predominant proinflammatory and catabolic cytokines involved in disease initiation and progression. Other proinflammatory cytokines may amplify or modulate this process, whereas anti-inflammatory cytokines, which are often detected in osteoarthritis tissues, may counteract the tissue destruction and inflammation. 
     Interleukin-1β plays a key role in the pathology of osteoarthritis or intervertebral disc generation/prolapse. The biological antagonist, interleukin-1 receptor antagonist (IL-1Ra), intervenes in the physiological mechanism of these diseases. 
     Systems and methods of producing serum and compositions with enhanced IL-1Ra are known in the art. For example, a system and method of producing autologous IL-1Ra in an efficient manner are disclosed in U.S. Pat. No. 8,460,227 issued Jun. 11, 2013, the disclosure of which is incorporated by reference in its entirety herewith. The RAP II device disclosed in U.S. Pat. No. 8,460,227 provides anti-inflammatory cytokines for treatment of human or non-human damaged tissue such as cartilage and neurological tissue, specifically IL-1Ra. Blood is withdrawn from the patient and then transferred into a special container provided with a cap configured to allow the patient&#39;s blood to flow on an inner side of the container, to ensure maximum contact with the increased surface area sides of the container. The container is then incubated and centrifuged. Subsequent to the incubation and the centrifugation, the serum containing autologous IL-1Ra is withdrawn and injected back into the patient. Although the serum obtained with the TRAP II device has a high concentration of interleukin-1 receptor antagonist (IL-1Ra), the serum also has a high concentration of interleukin 1-beta (IL-1β) and, thus, an overall low IL-1Ra/IL-1β ratio. 
     There is a need for devices and methods for specific protein capture to promote healing responses in vivo and healing of tissue injuries. Also needed are methods and devices for selectively removing (filtering out) unwanted components (products, byproducts and/or specific output of cells) from compositions such as, e.g., autologous fluid or serum. Systems and methods of producing serum that has a high concentration of interleukin-1 receptor antagonist (IL-1ra) and reduced interleukin IL-1β (IL-1β) so that the IL-1Ra inhibits the catabolic effects of IL-1β in vivo are also needed. A device that minimizes IL-1β delivered to the damaged tissue by selectively removing the cytokine and, thus, that increases the overall IL-1Ra/IL-1β ratio is also needed. 
     SUMMARY OF THE INVENTION 
     The present invention provides techniques and devices for removing (filtering out) unwanted/inhibitory components (for example, products, byproducts and/or cell output such as inhibitory catabolic proteins) from a composition (for example, autologous fluid or serum) containing such unwanted/inhibitory components. 
     The system of the present invention comprises at least one construct designed to contain a composition (for example, autologous fluid or serum) containing inhibitory/unwanted components (products, byproducts and/or output of cells such as inhibitory catabolic proteins). The construct is provided with interior walls including specific protein capturing means designed to remove the inhibitory/unwanted component(s) (for example, specific protein(s)) from the composition. Proteins targeted for capture include, but are not limited to, catabolic cytokines such as IL-1β, IL-α, IL-6, TNFα, IFNγ and MMPS as these proteins inhibit the healing process. 
     Other features and advantages of the present invention will become apparent from the following description of the invention, which refers to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1( a )  illustrates a first exemplary embodiment of a construct (vial) coated with specific protein/antibodies of a system of the present invention (catabolic cytokines removal system). 
         FIG. 1( b )  illustrates a second exemplary embodiment of a construct (tubing) coated with specific protein/antibodies of a system of the present invention (catabolic cytokines removal system). 
         FIG. 1( c )  illustrates a third exemplary embodiment of a construct (syringe) coated with specific protein/antibodies of a system of the present invention (catabolic cytokines removal system). 
         FIG. 2  illustrates the protein coated tubing of  FIG. 1( b )  attached to two exemplary syringes. 
         FIGS. 3( a ) and 3( b )  illustrate additional views of the constructs coated with specific protein/antibodies of the system of the present invention (RAP II MAX). 
         FIGS. 4( a ) and 4( b )  illustrate graphs with the levels of IL-1β when serum is incubated (soaked) in antibody coated devices ( FIG. 4( a ) ) and when serum is passed through a coated device ( FIG. 4( b ) ), and according to methods of the present invention (RAP II MAX). 
         FIGS. 5( a )-( c )  illustrate graphs showing that the IL-1Ra levels remained about the same after passing through anti-IL-1β antibodies. 
         FIGS. 6( a ) and 6( b )  illustrate graphs with the ratio of IL-1Ra/IL-1β in serum obtained with methods of the present invention (RAP II MAX). 
         FIG. 6( c )  illustrates a comparison of IRAP and IRAP II IL-1Ra/IL-1β ratios. 
         FIGS. 7( a ) and 7( b )  illustrate graphs with the levels of IL-1β and IL-1Ra when serum is obtained with IRAP II with collagen as incubation construct. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, reference is made to various specific embodiments in which the invention may be practiced. These embodiments are described with sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be employed, and that structural and logical changes may be made without departing from the spirit or scope of the present invention. 
     The present invention provides techniques and devices that prevent (minimize) the introduction of catabolic cytokines into damaged tissue and/or joint space by providing specific cytokine binding before introduction (injection). The systems of the present invention provide compositions with increased anabolic and anti-inflammatory cytokines for treatment of human or non-human damaged tissue such as cartilage and neurological tissue. 
     The system of the present invention comprises at least one construct designed to contain (hold) a composition (for example, serum or autologous fluid such as ACP, PRP, BMA, and BMC, among others). The construct is provided with interior walls including specific protein capturing means (binding site, for example, capture antibodies with certain protein targets) designed to remove specific protein(s) from the composition. Proteins targeted for capture include, but are not limited to, IL-1β, IL-α, IL-6, TNFα, IFNγ and MMPS as these proteins inhibit the healing process. 
     The present invention also provides methods for selectively removing (filtering out) unwanted/inhibitory components (products, byproducts and/or specific output of cells such as inhibitory catabolic proteins) from a specific composition (such as, e.g., autologous fluid or serum). The system is provided with a binding site for binding the unwanted/inhibitory component and removing such component from the composition. 
     In an exemplary-only embodiment, the systems and devices of the present invention bind IL-1β which plays a central role in the pathophysiology of cartilage damage and degradation in osteoarthritis. The system includes a construct (for example, a tube or vial such as a double ended tube or a capped/ribbed vial or syringe, among others) having interior walls coated with specific antibodies that are bound to the inside of the construct. When autologous fluid or serum is passed (filtered) through the construct or incubated in the construct, specific catabolic cytokines (e.g., IL-1β are bound and are selectively filtered out of the fluid (serum). In an exemplary-only embodiment, the walls are provided with specific antibodies (anti IL-1β antibodies) that remove IL-1β from serum obtained with an TRAP II system (such as the one disclosed in U.S. Pat. No. 8,460,227, the disclosure of which is incorporated in its entirety by reference herewith). In this manner, the system of the present invention minimizes IL-1β delivered to the damaged tissue by selectively binding the cytokine to the walls of the construct (tube or vial). 
     The present invention also provides a method of removing (filtering out) unwanted/inhibitory cell output present in an autologous fluid or serum (i.e., unwanted material produced by cells such as healing and growth inhibitory proteins) to clean the fluid (serum) and deliver the cleaned fluid (serum) to an injured site for treatment of connective tissue injuries. The method comprises the steps of (i) providing an apparatus comprising a container with walls supporting at least one protein capturing means (binding site); (ii) introducing serum (fluid) into the container; (iii) allowing the serum to interact with the protein capturing means, so that inhibitory unwanted proteins are captured by (bound to) the protein capturing means and forming a cleaned fluid (serum); and (iv) removing the cleaned serum containing less inhibitory proteins (inhibitory healing proteins) from the container. 
     The present invention also provides a method of providing a serum with minimized IL-1β (and increased IL-1Ra/IL-1β ratio) delivered to a damaged tissue by selectively removing the IL-1β from a concentrated serum. The method comprising the steps of (i) providing a device (tube or vial) with internal walls coated with anti-IL-1β antibodies so that the anti-IL-1β antibodies are bound to the inner surface of the device; (ii) passing RAP II serum through the device or injecting/pipetting the serum into the device; (iii) capturing the IL-1β from the serum with the coated anti-IL-1β antibodies, and removing IL-1β from the serum; and (iv) removing the cleaned serum containing less inhibitory proteins (inhibitory healing proteins), and injecting the removed serum into a patient. The system of the present invention acts as a filter to remove unwanted/inhibitory material(s) and/or byproducts produced by cells. 
     Referring now to the drawings, where like elements are designated by like reference numerals,  FIGS. 1( a )-3( b )  illustrate exemplary embodiments of a device  10   a ,  10   b ,  10   c  of system  100  of the present invention designed to target and capture a specific protein (or multiple proteins), to promote healing responses in vivo. 
     Devices 
     The devices  10   a ,  10   b ,  10   c  may have any shape/configuration and may be formed of materials such as plastics with protein binding capabilities, for example polystyrene, PVC, PETG or PC, etc. Plastics without protein binding capabilities such as polyolefins (for example, PP) are undesirable. The device/construct of the present invention may be any tube, vial, jar, syringe, tubing, etc. that can be sterilized capped, and/or closed, and that can hold any composition (for example, serum or autologous fluid such as ACP, PRP, BMA, and BMC, among many others). 
       FIGS. 1( a )-( c )  show devices  10   a ,  10   b ,  10   c  provided with one or more internal surfaces on the interior walls having formations that enlarge the surface area, for example, in the form of internal ribbing  22 . The internal ribbing creates more surface area within the construct for more protein binding. Construct  10   a  may be any tube, vial, jar, etc. Construct  10   b  is an exemplary tube/tubing with both ends open. Construct  10   c  is an exemplary syringe with internal ribbing  22  provided with a protein binding site. Constructs  10   a ,  10   b ,  10   c  are all provided with internal walls and/or structures supporting specific protein/antibody  50  (capture antibodies  50 ). Each construct is coated with specific protein/antibody and serum can be incubated in the container or allowed to pass through the container (in the case of a tubing) back and forth, or just one full time. 
     In an exemplary embodiment, tubing construct  10   b  of  FIG. 1( b )  is provided with walls supporting specific protein/antibody  50  (capture antibodies  50 ). In an exemplary embodiment, tubing construct  10   b  is coated with the specific protein/antibody  50 . Subsequent to the coating of device  10   b , serum (for example, concentrated serum or fluid such as IRAP II serum) can be passed back and forth, or just once trough the tubing, from a syringe on one side of the tubing to a syringe on the other side of the tubing (as shown in  FIG. 2 ). 
     System  100  of  FIG. 2  illustrates device  10   b  (protein coated tubing  10   b ) connected to a first syringe  21  (containing original non-treated serum  80 ) and to a second syringe  23  (for collecting the protein coated-tubing “treated” serum  88 ). Serum  80  (for example, an TRAP II serum) is pushed through the first syringe  21  in the direction of arrow A, passed through a first valve  31  into the protein coated tubing  10   b  and then through second valve  33  into the second syringe  22 , and out of the syringe  22  in the direction of arrow B ( FIG. 2 ). The obtained TRAP II MAX serum  88  may be further removed and injected into the body (at the specific surgical site of interest) or frozen for further injection/application. 
       FIGS. 3( a ) and 3( b )  illustrate additional views of the constructs coated with specific protein/antibodies of the system of the present invention (RAP II MAX).  FIG. 3( a )  shows capped vial  10   a ″ which includes vial  10   a  of  FIG. 1( a )  with cap  13  and serum incubated therein to allow binding of IL-1β to the specific protein site  50  formed on the inner walls of the vial  10   a .  FIG. 3( b )  illustrates capped vial  10   a ′ which includes vial  10   a  of  FIG. 1( a )  with cap  13  and without serum, and also tubing  10   b ′ which include tubing  10   b  of  FIG. 1( b )  with a cap  13  at each end. 
     In the above-described embodiments, anti-IL-1β antibodies bound the inner surface of the device. The serum is passed through the tube. The serum is incubated in container. The IRAP II serum may be incubated in the container for a set amount of time and then it can be removed and frozen for injection. Alternatively, the IRAP II serum may be passed through the tube (tubing) and then frozen for further application/injection. 
     Coating of device constructs (such as devices  10   a ,  10   b ,  10   c  of  FIGS. 1( a )-( c ) ) are conducted with a specific protein/antibody to capture a specific protein within serum (protein binding affinity). The specific protein/antibody is coated on at least a portion of the inner wall(s) of the devices. Proteins targeted for capture include, but are not limited to, IL-1β, IL-α, IL-6, TNFα, IFNγ and MMPS as these proteins inhibit the healing process. 
     Capture antibodies with certain protein targets  50  are used in a predetermined concentration to incubate within the device construct(s)  10   a ,  10   b ,  10   c  for a set time interval at room temperature. After the incubation period, once the capture antibodies  50  have had time to bind to the device construct(s)  10   a ,  10   b ,  10   c , the device construct(s) are rinsed to remove excess proteins that have not bound. The device construct(s)  10   a ,  10   b ,  10   c  (coated with capture antibodies with certain protein targets  50 ) are then stored in a refrigerator. 
     Procedure 
     Serum  80  (such as TRAP II serum  80 ) is injected/pipetted into the antibody coated device construct(s)  10   a ,  10   b ,  10   c . The serum is then allowed to sit in these antibody coated devices  10   a ,  10   b ,  10   c  in a refrigerator and for a set time period. After the serum is allowed to sit in the coated device(s)  10   a ,  10   b ,  10   c , cleaned/treated serum  88  is removed and made available for injection. 
     Outcome 
     Serum  80  ( FIG. 2 ) will interact with the capture antibodies coating the device construct(s)  10   a ,  10   b ,  10   c . The unwanted proteins within the serum which cause adverse effects to the healing process will be captured or bound by the antibody coating  50  on the inside of the device construct(s)  10   a ,  10   b ,  10   c . The serum  88  removed from the coated device construct(s)  10   a ,  10   b ,  10   c  will contain less inhibitory protein (specific to the capture antibody or antibodies) because most will be bound to the capture antibodies coating the device(s)  10   a ,  10   b ,  10   c.    
     The devices and techniques detailed above consist of either a double ended tube  10   b , or a capped/ribbed vial  10   a ,  10   c  that autologous fluid can be filtered through. What makes the tube or vial unique is the coating of specific antibodies  50  that is bound to the inside of the tube. When serum is passed through the tube or incubated in the vial, specific cytokines are bound and selectively filtered out of the fluid/serum. 
     In an exemplary-only embodiment, devices  10   a ,  10   b ,  10   c  specifically utilize the output serum  80  from the IRAP II device. However, the systems and techniques of the present invention are not limited to serum, and can be implemented with different proteins and cytokines from autologous fluids like ACP, PRP, BMA, and BMC, among others. The serum is injected into a tube or vial which has been coated with anti-bodies specific for IL-1β. When the serum passes through—or is injected into—the device (tube or vial or tube/vial containing an anti-body substrate), the IL-1β is taken out of solution. The ratio IL-1Ra/IL-1β becomes higher and the resulting serum  88  can be injected directly into, on top, or around the damaged tissue site. 
     Reference is now made to  FIGS. 4( a ) and 4( b )  which illustrate graphs with the levels of IL-1β when serum is incubated (soaked) in antibody coated devices ( FIG. 4( a ) ) and when serum is passed through a coated device ( FIG. 4( b ) ), and according to methods of the present invention (with the IRAP II MAX system and method). Levels of IL-1β were reduced whether incubating the serum in coated device or passing the serum through a coated tube.  FIG. 4( a )  shows that the IL-1β levels were reduced between 50 and 60% using the coated construct.  FIG. 4( b )  shows that the IL-1β levels were reduced by about 60% by passing through the coated tube. 
       FIGS. 5( a )-( c )  illustrate graphs showing that the IL-1Ra levels of the IRAP II MAX system and method remained about the same after passing through anti-IL-1β antibodies.  FIG. 5( a )  shows the IL-1Ra levels relative to various concentrations of antibody coated tubes (i.e., 5 μg/mL, 10 μg/mL and 25 μg/mL).  FIG. 5( b )  shows the different concentrations of the antibody coated tubes, 5 μg/mL, 10 μg/mL and 25 μg/mL.  FIG. 5( c )  shows that the IL-1Ra levels remain about the same regardless of the number of passes through the antibody coated tubing (an exemplary PVC antibody coated tubing). 
       FIGS. 6( a ) and 6( b )  illustrate two graphs with the ratio of IL-1Ra/IL-1β in serum obtained with methods of the present invention (IRAP II MAX system and methods), i.e., by soaking/incubating in a coated device at various antibody concentrations ( FIG. 6( a ) ) and by passing through a PVC antibody coated device between 0-25 passes ( FIG. 6( b ) ).  FIG. 6( c )  illustrates a comparison of IRAP and IRAP II IL-1Ra/IL-1β ratios. Due to no change in the IL-1Ra concentration and the reduction in the IL-1β concentration, the IL-1Ra/IL-1β ratio increases up to four times (about 4× fold). 
       FIGS. 7( a ) and 7( b )  illustrate graphs with the levels of IL-1β and IL-1β when serum is obtained with IRAP II with collagen as incubation construct.  FIG. 7( a )  shows high IL-1Ra concentrations; however,  FIG. 7( b )  also shows high IL-1β concentrations. Using an IRAP II MAX system of the present invention would greatly reduce the IL-1β levels while the IL-1Ra elevated levels would remain still elevated. The IRAP II MAX is the IRAP II with collagen or biocartilage+IL-1β filter. 
     The systems and methods of the present invention (IRAP II MAX) provide the following advantages:
         IL-1Ra/IL-1β ratios can be maximized by selectively filtering IRAP II serum with constructs coated with anti-IL-1β (4× increase);   IRAP II MAX could be created by incubating blood with collagen or bio-cartilage, then selectively filtering with anti-IL-1β;   this technique requires a minimal amount of passes through the tube to maximize IL-1β removal;   this technique allows coating of the device (tube), then packaging, labeling and sterilizing the coated device (tube), and providing the coated device in assembled (or pre-assembled) form, with overall decreased manufacturing costs; and   this technique may be useful for selectively filtering out unwanted WBC with bone marrow concentrate using different anti-antibodies.       

     The techniques and devices of the present invention prevent/minimize the injection of catabolic cytokines into damaged tissue and/or joint space by specific cytokine binding before injection. The devices and systems of the present invention bind IL-1β which plays a central role in the pathophysiology of cartilage damage and degradation in osteoarthritis. The IRAP II device (such as the one disclosed in U.S. Pat. No. 8,460,227) produces serum that has a high concentration of interleukin 1-beta receptor antagonist (IL-1Ra) and interleukin IL-1β (IL-1β). The IL-1Ra inhibits the catabolic effects of IL-1β in vivo. The IRAP II MAX device and system of the present invention minimizes IL-1β delivered to the damaged tissue by selectively binding the cytokine to the walls of the tube or vial. The IRAP II MAX device of the present invention uses the output from the IRAP II device (the serum) and filters it accordingly. When serum or fluid (for example, serum from IRAP II) is passed through the tube or incubated in the vial, specific cytokines are bound and selectively filtered out of the fluid/serum. 
     While the present invention is described herein with reference to illustrative embodiments for particular applications, it should be understood that the invention is not limited thereto. Those having ordinary skill in the art and access to the teachings provided herein will recognize additional modifications, applications, embodiments and substitution of equivalents all fall within the scope of the invention.