Functionalized Erythroid Cells

Abstract

Described herein are novel preparations of functionalized erythroid cells and related compositions, reagents, and methods for use in human pharmaceutical and veterinary applications.

What is claimed is: 
     
         1 . An enucleated erythroid cell, comprising an exogenous polypeptide agent covalently linked to the cell surface by a residual linker comprising a click signature, via an amino acid side chain of a protein at the cell surface, wherein the click signature was formed as the product of a click reaction or has the structure of a click signature. 
     
     
         2 . An enucleated erythroid cell, comprising:
 a plurality of exogenous polypeptide agents, each exogenous polypeptide agent of the plurality being covalently linked to the cell surface by a residual linker comprising a click signature,   wherein at least 10% of exogenous polypeptide agents on the cell are linked via an amino acid side chain to a protein at the cell surface,   wherein the click signature was formed as the product of a click reaction or has the structure of a click signature.   
     
     
         3 . The enucleated erythroid cell of  claim 2 , wherein at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of exogenous polypeptide agents on the cell are linked via an amino acid side chain to a protein at the cell surface. 
     
     
         4 . The enucleated erythroid cell of any of the preceding claims, which is not genetically engineered. 
     
     
         5 . The enucleated erythroid cell of any of the preceding claims, which does not comprise a non-natural amino acid. 
     
     
         6 . The enucleated erythroid cell of any of the preceding claims, wherein the exogenous polypeptide agent comprises a n-clamp. 
     
     
         7 . The enucleated erythroid cell of  claim 6 , wherein the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell via the π-clamp. 
     
     
         8 . The enucleated erythroid cell of any of the preceding claims, wherein the exogenous polypeptide agent comprises a non-canonical amino acid (ncAA). 
     
     
         9 . The enucleated erythroid cell of  claim 8 , wherein the exogenous polypeptide agent is covalently linked to the enucleated erythroid cell via the ncAA. 
     
     
         10 . The enucleated erythroid cell of any of the preceding claims, which lacks a sortase transfer signature. 
     
     
         11 . The enucleated erythroid cell of claim any of the preceding claims, wherein the exogenous polypeptide agent is selected from an alpha4beta7 antibody, IL10, an enzyme such as asparaginase, or a clotting factor such as Factor Xa. 
     
     
         12 . The enucleated erythroid cell of any of the preceding claims, which comprises at least about 5,000, 10,000, 50,000, 100,000, or 200,000 copies of the exogenous polypeptide agent, or between 5,000-10,000, 10,000-50,000, 50,000-100,000, or 100,000-200,000 copies of the exogenous polypeptide agent. 
     
     
         13 . The enucleated erythroid cell of any of the preceding claims, wherein the exogenous polypeptide agent is a peptide ligand that binds a binding partner. 
     
     
         14 . The enucleated erythroid cell of any of the preceding claims, which has a clearance rate wherein at least 20% of the agent remains in the circulatory system of the subject over 1, 2, 3, 4, 5, 6, or 7 days. 
     
     
         15 . The enucleated erythroid cell of any of the preceding claims, wherein less than 10% of coupling reagent on the cell is unreacted coupling reagent. 
     
     
         16 . The enucleated erythroid cell of any of the preceding claims, which further comprises a second exogenous polypeptide agent, e.g., wherein the second exogenous polypeptide agent is covalently linked to the cell surface by a second residual linker comprising a click signature. 
     
     
         17 . The enucleated erythroid cell of  claim 16 , which further comprises a third exogenous polypeptide agent, e.g., wherein the third exogenous polypeptide agent is covalently linked to the cell surface by a second residual linker comprising a click signature. 
     
     
         18 . The enucleated erythroid cell of any of the preceding claims, wherein at least 50%, 60%, 70%, 80%, or 90% of the exogenous polypeptide agents are covalently linked to the cell surface with a preselected orientation, e.g., are attached by the same moiety of the exogenous polypeptide agents (e.g., an N-terminus, a C-terminus, or a particular residue, e.g., a cysteine). 
     
     
         19 . The enucleated erythroid cell of any of the preceding claims, wherein the exogenous polypeptide agent is covalently linked to an endogenous molecule of the cell, e.g., an endogenous polypeptide at the cell surface. 
     
     
         20 . A preparation comprising at least 10 10 , 10 11 , or 10 12  enucleated erythroid cells of any of the preceding claims. 
     
     
         21 . The preparation of  claim 20 , wherein at least 70% of the enucleated erythroid cells in the preparation comprise the agent. 
     
     
         22 . The preparation of  claim 20  or  21  wherein at least 70% of the enucleated erythroid cells in the preparation comprise an exogenous polypeptide agent that binds a ligand, e.g., in a flow cytometry assay of Example 6. 
     
     
         23 . The preparation of any of  claims 20 - 22  wherein at least 50%, 60%, 70%, 80%, or 90% of the exogenous polypeptide agents in the preparation are covalently linked to an enucleated erythroid cell surface with a preselected orientation, e.g., are attached by the same moiety of the exogenous polypeptide agents (e.g., an N-terminus, a C-terminus, or a particular residue, e.g., a cysteine). 
     
     
         24 . A method of making an enucleated erythroid cell functionalized with an agent comprising:
 (a) providing an activated cell comprising a cell covalently bound to a first click handle,   (b) providing an activated agent comprising an agent (e.g., an exogenous polypeptide) covalently bound to a second click handle capable of reacting with the first click handle, and   (c) contacting the activated cell with the activated agent, thereby producing a cell functionalized with the agent.   
     
     
         25 . An enucleated erythroid cell produced by the method of  claim 24 . 
     
     
         26 . A method of administering an agent to a human subject, comprising administering an effective number of cells of any of  claims 1 - 19  to the human subject, thereby administering the agent to the human subject. 
     
     
         27 . A cell of any of  claims 1 - 19  for use as a medicament.