Therapeutic protein compositions and methods of making and using the same

Abstract

Disclosed herein are compositions and methods for preparation and use of protein therapeutics, and more particularly protein clusters or backpacks having a plurality of therapeutic protein monomers reversibly crossed-linked by biodegradable linkers.

1 - 25 . (canceled) 
     
     
         26 . A method for preparing a therapeutic composition the method comprising reacting a plurality of therapeutic protein monomers with A plurality of biodegradable cross-linkers, thereby cross-linking the therapeutic protein monomers into a protein cluster,
 wherein the protein cluster has a size between 30 nm and 1000 nm in diameter measured by dynamic light scattering,   wherein the cross-linker has a formula of formula (II):   
       
         
           
           
               
               
           
         
       
       wherein:
 X 1  and X 2  are each independently selected from the group consisting of triflyl, tosyl, and N-succinimidyl: 
 A 1  and A 3  are both —(CH 2 ) 2 —; 
 A 2  is —(CR 1 R 2 ) m —; 
 Y 1  and Y 2  are both O; 
 wherein R 1  and R 2  at each occurrence are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  cycloalkyl, C 2-12  heterocyclyl, C 6-12  aryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl, and C 4-12  heteroaryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl: and 
 m is an integer selected from 0-12, and 
 wherein the cross-linker portion of the protein cluster degrades, after administration into a subject in need thereof, under physiological conditions so as to release the therapeutic protein monomers from the protein cluster. 
 
     
     
         27 . The method of  claim 26 , wherein the reacting step is performed at a temperature between about 5° C. and about 40° C. 
     
     
         28 . The method of  claim 26 , wherein the reacting step is performed for about 1 minute to about 8 hours. 
     
     
         29 . The method of  claim 26 , further comprising providing the surface modification to the protein cluster. 
     
     
         30 . The method of  claim 26 , further comprising purifying the protein cluster. 
     
     
         31 . A method for preparing a cell therapy composition, comprising:
 providing a therapeutic composition comprising (i) a protein cluster comprising a plurality of therapeutic protein monomers reversibly cross-linked to one another and (ii) a pharmaceutically acceptable carrier or excipient; and   incubating the protein cluster with a nucleated cell preferably for about 30-60 minutes,
 wherein the protein cluster has a size between 30 nm and 1000 nm in diameter measured by dynamic light scattering and is produced by reacting the plurality of therapeutic protein monomers with a plurality of biodegradable cross-linkers of formula (II): 
   
       
         
           
           
               
               
           
         
       
       wherein:
 X 1  and X 2  are each independently selected from the group consisting of triflyl, tosyl, and N-succinimidyl; 
 A 1  and A 3  are each independently —(CR 1 R 2 ) n  —: 
 A 2  is —(CR 1 R 2 ) m ; 
 Y 1  and Y 2  are each independently selected from NR 3 , O and S: 
 R 1  and R 2  at each occurrence are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  cycloalkyl, C 2-12  heterocyclyl, C 6-12  aryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl, and C 4-12  heteroaryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl: 
 wherein R 3  is selected from the group consisting of hydrogen, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  cycloalkyl, C 2-12  heterocyclyl, C 6-12  aryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl, and C 4-12 heteroaryl optionally substituted with 1 or more halo: hydroxyl: 
 C 1-6  alkyl and/or C 1-6  alkoxyl: 
 n, at each occurrence, is an integer independently selected from 1-12: and 
 m is an integer selected from 0-12, 
 thereby cross-linking the therapeutic protein monomers into the protein cluster, wherein the cross-linker portion of the protein cluster degrades, after administration into a subject in need thereof, under physiological conditions so as to release the therapeutic protein monomers from the protein cluster. 
 
     
     
         32 . A cell therapy composition, comprising a therapeutic composition comprising a protein cluster comprising (i) a plurality of therapeutic protein monomers reversibly cross-linked to one another and (ii) a pharmaceutically acceptable carrier or excipient, associated with a nucleated cell,
 wherein the protein cluster has a size between 30 nm and 1000 nm in diameter measured by dynamic light scattering and is produced by reacting the plurality of therapeutic protein monomers with a plurality of biodegradable cross-linkers of formula (II):   
       
         
           
           
               
               
           
         
       
       wherein:
 X 1  and X 2  are each independently selected from the group consisting of triflyl, tosyl, and N-succinimidyl: 
 A 1  and A 3  are each independently —(CR 1 R 2 ) n —: 
 A 2  is —(CR 1 R 2 ) m —; 
 Y 1  and Y 2  are each independently selected from NR 3 , O and S: 
 R 1  and R 2  at each occurrence are independently selected from the group consisting of hydrogen, halogen, hydroxyl, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  cycloalkyl, C 2-12  heterocyclyl, C 6-12  aryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl, and C 4-12  heteroaryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl: and 
 wherein R 3  is selected from the group consisting of hydrogen, C 1-12  alkyl, C 2-12  alkenyl, C 3-12  cycloalkyl, C 2-12  heterocyclyl, C 6-12  aryl optionally substituted with 1 or more halo: hydroxyl: C 1-6  alkyl and/or C 1-6  alkoxyl, and C 4-12 heteroaryl optionally substituted with 1 or more halo: hydroxyl: 
 C 1-6  alkyl and/or C 1-6  alkoxyl: 
 n, at each occurrence, is an integer independently selected from 1-12: and 
 m is an integer selected from 0-12, 
 thereby cross-linking the therapeutic protein monomers into the protein cluster, 
 wherein the cross-linker portion of the protein cluster degrades, after administration into a subject in need thereof, under physiological conditions so as to release the therapeutic protein monomers from the protein cluster. 
 
     
     
         33 . A method of providing cell therapy, comprising administering the cell therapy composition of  claim 32  into a subject in need thereof. 
     
     
         34 . The method of  claim 26 , wherein the cross-linker is symmetrical. 
     
     
         35 . The method of  claim 26 , wherein X 1  and X 2  are both N-succinimidyl. 
     
     
         36 . The method of  claim 26 , wherein R 1  and R 2  are both hydrogen. 
     
     
         37 . The method of  claim 26 , wherein A 2  is —(CH 2 ) 2 —. 
     
     
         38 . The method of  claim 26 , wherein the cross-linker is: 
       
         
           
           
               
               
           
         
       
     
     
         39 . The method of  claim 26 , wherein A 2  is a bond. 
     
     
         40 . The method of  claim 26 , wherein the therapeutic protein monomers comprise one or more cytokine molecules selected from the group consisting of IL-15, IL-2, IL-7, IL-10, IL-12, IL-18, IL-21, IL-23, IL-4, IL-lα, IL-lβ, IL-5, IFNγ, TNFα, IFNα, IFNβ, GM-CSF, and GCSF. 
     
     
         41 . The method of  claim 26 , wherein the therapeutic protein monomers comprise IL-15. 
     
     
         42 . The method of  claim 26 , wherein the protein cluster further comprises a polycation on its surface.