Polypeptide compositions comprising spacers

Abstract

Disclosed herein are methods and compositions including antigen-binding polypeptides comprising a stalk region and a stalk extension region. In some cases, the antigen-binding compositions comprising the stalk extension region has increased expression on a cell surface and, in some cases, has increased antigen-binding efficiency. A subject antigen binding polypeptide can be a chimeric antigen receptor (CAR).

1 . (canceled) 
     
     
         2 . The method of  claim 85 , wherein the stalk region is from about 20 to about 60 amino acids in length and the spacer region comprises from 1 to 5 stalk extension regions with each stalk extension region comprising fewer dimerization sites than the stalk region. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . The method of  claim 85 , wherein the stalk region is proximal to the membrane region and the stalk extension region lacks a dimerization site. 
     
     
         6 - 13 . (canceled) 
     
     
         14 . The method of  claim 85 , wherein the stalk extension region comprises an amino acid sequence having at least 80% identity with the amino acid sequence of the stalk region. 
     
     
         15 - 19 . (canceled) 
     
     
         20 . The method of  claim 85 , wherein the stalk region comprises a sequence with at least about 80% identity to a CD8alpha hinge domain, a CD28 hinge domain, or a CTLA-4 hinge domain. 
     
     
         21 . The method of  claim 85 , the stalk region comprises an amino acid sequence having at least 80% identity SEO ID NO: 3. 
     
     
         22 - 26 . (canceled) 
     
     
         27 . The method of  claim 85 , wherein the antigen binding region binds an epitope on CD19, BCMA, CD44, α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, Folate receptor α, a mucin, GPC3, CSPG4, HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP, c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2, CLL-1, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123, and/or VEGF-R2. 
     
     
         28 . The method of  claim 85 , wherein the chimeric polypeptide comprises a chimeric antigen receptor (CAR). 
     
     
         29 - 30 . (canceled) 
     
     
         31 . The method of  claim 85 , wherein the chimeric polypeptide further comprises a signaling domain from 4-1BB, CD28, or a combination thereof. 
     
     
         32 . The method of  claim 85 , wherein the chimeric polypeptide further comprises a CD3-zeta signaling domain. 
     
     
         33 - 43 . (canceled) 
     
     
         44 . The method of  claim 85 , wherein the cell further comprises a Sleeping Beauty transposase. 
     
     
         45 . The method of  claim 44 , wherein the Sleeping Beauty transposase is SB11, SB100X or SB110. 
     
     
         46 - 47 . (canceled) 
     
     
         48 . The method of  claim 85 , wherein the cell is a human T cell or NK cell. 
     
     
         49 - 68 . (canceled) 
     
     
         69 . The method of  claim 85 , wherein the chimeric polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 53-68, or a functional variant thereof having at least 80% sequence identity therewith. 
     
     
         70 - 84 . (canceled) 
     
     
         85 . A method for treating a hyperproliferative disorder, the method comprising administering to a subject in need thereof a cell expressing a chimeric polypeptide comprising:
 (a) an antigen binding region;   (b) a transmembrane region; and   (c) a spacer region connecting the transmembrane and antigen binding regions, wherein the spacer region comprises: (i) a stalk region comprising at least one dimerization site; and (ii) a stalk extension region comprising fewer dimerization sites than the stalk region.   
     
     
         86 . The method of  claim 85 , wherein the antigen binding domain binds an epitope on CD19, CD33, ROR1, mesothelin, CD22, and/or MUC-16. 
     
     
         87 . The method of  claim 85 , wherein the antigen binding region binds an epitope on CD19, CD33, ROR1, and/or EGFR. 
     
     
         88 . The method of  claim 85 , wherein the antigen binding region binds an epitope on ROR1. 
     
     
         89 . The method of  claim 85 , wherein the cell is administered to the subject without undergoing propagation and activation. 
     
     
         90 . The method of  claim 85 , wherein the spacer region comprises an amino acid sequence having at least 80% identity with SEQ ID NO: 5. 
     
     
         91 . The method of  claim 85 , wherein the spacer region comprises an amino acid sequence having the sequence of SEQ ID NO: 5.