Synovial extracellular matrix-specific chimeric antigen receptor for targeting regulatory t cells to treat autoimmune diseases

Abstract

Disclosed herein are chimeric antigen receptors (“CARs”) comprising an antigen binding site that recognizes citrullinated polypeptides. Citrullinated polypeptides, such as citrullinated vimentin, fibrinogen, and filaggrin, are expressed in the synovium of subjects with rheumatoid arthritis. Further disclosed are T cells, and in particular, Treg cells, that express these CARs. Administration of these CAR-T cells is useful in the treatment of rheumatoid arthritis as well as other diseases associated with citrullinated peptides.

1 - 43 . (canceled) 
     
     
         44 . A kit comprising a container containing a pharmaceutical composition communicating through a fluidic conduit with a drip chamber, wherein the drip chamber communicates through a fluidic conduit with an intravenous needle, and wherein
 the pharmaceutical composition comprising a plurality of Treg cells engineered to express a chimeric antigen receptor (CAR-Treg), and a pharmaceutically acceptable carrier,   the CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain,
 the antigen binding domain binds to citrullinated vimentin, citrullinated filaggrin, and citrullinated fibrinogen, or citrullinated fragments thereof, and 
 the antigen binding domain comprises a VH domain and a VL domain, wherein:
 (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and 
 (ii) the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:43. 
 
   
     
     
         45 . The kit of  claim 44 , wherein the container comprises a bag. 
     
     
         46 . The kit of  claim 44 , wherein the fluidic conduit between the container and the needle comprises one or more Y-sites and a roller clamp. 
     
     
         47 . A method of preparing T regulatory cells (Treg) expressing a chimeric antigen receptor (CAR), the method comprising:
 (a) isolating T cells from a biological sample obtained from a human subject;   (b) enriching the T cells for T regulatory cells (Treg);   (c) transfecting the enriched Treg cells with an expression vector encoding the CAR to produce CAR-Treg cells; and   (d) expanding the CAR-Treg cells to produce a plurality of CAR-Treg cells, wherein   the CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain,   the antigen binding domain binds to citrullinated vimentin, citrullinated filaggrin, and citrullinated fibrinogen, or citrullinated fragments thereof, and   the antigen binding domain comprises a VH domain and a VL domain, wherein:
 (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and 
 (ii) the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:43. 
   
     
     
         48 . The method of  claim 47 , wherein the expansion comprises using anti-CD3/CD28 coated beads. 
     
     
         49 . The method of  claim 47 , wherein the expansion does not comprise using anti-CD3/CD28 coasted beads. 
     
     
         50 . The method of  claim 47 , wherein the transfection occurs by use of a viral vector, electroporation, heat shock, bacteriophage, sonication, or calcium phosphate. 
     
     
         51 . The method of  claim 50 , wherein the transfection occurs by use of a viral vector. 
     
     
         52 . The method of  claim 47 , wherein the antigen-binding domain comprises a single chain variable fragment (scFv) comprising:
 (a) a VH domain comprising the amino acid sequence having at least 95% identity to SEQ ID NO:1; and   (b) a VL domain comprising the amino acid sequence having at least 95% identity to SEQ ID NO:4.   
     
     
         53 . The method of  claim 52 , wherein the scFv comprises the amino acid sequence having at least 95% identity to SEQ ID NO:5. 
     
     
         54 . The method of  claim 47 , wherein the human subject has rheumatoid arthritis. 
     
     
         55 . A pharmaceutical composition comprising the plurality of the CAR-Treg cells produced by the method of  claim 54 , and a pharmaceutically acceptable carrier. 
     
     
         56 . A method of treating rheumatoid arthritis, the method comprising: administering the to the subject an effective amount of the pharmaceutical composition of  claim 55 . 
     
     
         57 . The method of  claim 56 , further comprising administering one or more anti-inflammatory and/or therapeutic agents to the subject. 
     
     
         58 . The method of  claim 57 , wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof. 
     
     
         59 . A method of treating rheumatoid arthritis, the method comprising administering to a subject an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier and a plurality of T regulatory cells engineered to express a chimeric antigen receptor (CAR), wherein
 the CAR comprises an antigen-binding domain, a hinge domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain,   the antigen binding domain binds to citrullinated vimentin, citrullinated filaggrin, and citrullinated fibrinogen, or citrullinated fragments thereof, and   the antigen binding domain comprises a VH domain and a VL domain, wherein:
 (i) the VH domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:32, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:34, and a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:36, and 
 (ii) the VL domain of the target-binding domain comprises a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:39, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:43. 
   
     
     
         60 . The method of  claim 59 , wherein the antigen-binding domain comprises a single chain variable fragment (scFv) comprising:
 (a) a VH domain comprising the amino acid sequence having at least 95% identity to SEQ ID NO:1; and   (b) a VL domain comprising the amino acid sequence having at least 95% identity to SEQ ID NO:4.   
     
     
         61 . The method of  claim 61 , wherein the scFv comprises the amino acid sequence having at least 95% identity to SEQ ID NO:5. 
     
     
         62 . The method of  claim 59 , further comprising administering one or more anti-inflammatory and/or therapeutic agents to the subject. 
     
     
         63 . The method of  claim 62 , wherein the anti-inflammatory agent is an anti-TNF antibody, an anti-IL-6 antibody, or a combination thereof.