Polypeptide compositions comprising spacers
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. A chimeric polypeptide comprising:
(a) an antigen-binding region;
(b) a transmembrane region; and
(c) a spacer region connecting the trans-membrane region with the antigen binding region, wherein the spacer region comprises: (i) a stalk region comprising about 20 to about 60 amino acids and at least one dimerization site; and (ii) one to five stalk extension regions, wherein each stalk extension region comprises fewer dimerization sites than the stalk region.
2. The chimeric polypeptide of claim 1 , wherein at least one stalk extension region comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of the stalk region.
3. The chimeric polypeptide of claim 1 , wherein the stalk region is proximal to the transmembrane region.
4. The chimeric polypeptide of claim 1 , wherein the stalk region is distal to the transmembrane region.
5. The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide exhibits increased expression on a cell surface compared to an otherwise identical polypeptide that lacks the stalk extension region.
6. The chimeric polypeptide of claim 1 , wherein at least one stalk extension region lacks a dimerization site.
7. The chimeric polypeptide of claim 1 , wherein each stalk extension region comprises about 20 to about 60 amino acids.
8. The chimeric polypeptide of claim 7 , wherein each stalk extension region comprises an amino acid sequence that has at least about 80% sequence identity to the amino acid sequence of the stalk region.
9. The chimeric polypeptide of claim 1 , wherein the stalk region comprises a sequence with at least about 80% identity to a CD8alpha hinge domain, a CD28 hinge domain, and/or a CTLA-4 hinge domain.
10. The chimeric polypeptide of claim 1 , 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, Mucins, MUC-1, MUC-16, 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.
11. The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide comprises a chimeric antigen receptor (CAR).
12. The chimeric polypeptide of claim 1 , wherein the chimeric polypeptide comprises an engineered T-cell receptor (TCR).
13. The chimeric polypeptide of claim 12 , wherein the engineered TCR is an αβ TCR.
14. The chimeric polypeptide of claim 12 , wherein the engineered TCR is a γδ TCR.
15. The chimeric polypeptide of claim 12 , wherein the antigen binding region binds an epitope on at least one of NY-ESO-1, Titin, MART-1, HPV, HBV, MAGE-A4, MAGE-A10, MAGE A3/A6, gp100, MAGE-A1, or PRAME.
16. The chimeric polypeptide of claim 11 , wherein at least one stalk extension region comprises: (a) an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of the stalk region; and (b) at least one amino acid substitution relative to the stalk region.
17. The chimeric polypeptide of claim 16 , wherein the stalk region is capable of dimerizing with a second stalk region present in a second CAR and comprises at least 80% sequence identity with the amino acid sequence of the second stalk region.
18. A method of increasing expansion of an engineered T cell expressing a chimeric polypeptide comprising engineering a nucleic acid encoding the chimeric polypeptide to comprise a stalk extension domain, thereby generating an engineered T cell.
19. The chimeric polypeptide of claim 1 , wherein the antigen binding region binds an epitope on CD33, MUC-16, and/or ROR1.
20. The chimeric polypeptide of claim 1 , wherein the antigen binding region binds an epitope on ROR1.
21. The chimeric polypeptide of claim 1 , wherein the stalk region comprises an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 3.
22. The chimeric polypeptide of claim 1 , wherein the spacer region comprises an amino acid sequence that has at least 80% sequence identity with SEQ ID NO: 5.
23. A modified immune effector cell that expresses the chimeric polypeptide of claim 1 .
24. A modified immune effector cell that expresses the chimeric polypeptide of claim 20 .
25. The modified immune effector cell of claim 24 , wherein the cell is a CAR-T cell.
26. The modified immune effector cell of claim 23 , wherein the cell expresses a cytokine.
27. The modified immune effector cell of claim 26 , wherein the cytokine is a membrane-bound IL15.
28. The modified immune effector cell of claim 23 , wherein the cell expresses a cell tag.
29. The modified immune effector cell of claim 28 , wherein the cell tag comprises a truncated epidermal growth factor receptor.
30. The modified cell of claim 23 , wherein the cell expresses a membrane-bound IL15 and a cell tag comprising a truncated epidermal growth factor receptor.
31. The modified cell of claim 24 , wherein the cell expresses a membrane-bound IL15 and a cell tag comprising a truncated epidermal growth factor receptor.
32. The modified cell of claim 23 , wherein the cell has improved cytotoxicity as compared to a cell expressing an otherwise identical antigen-binding polypeptide that lacks the stalk extension region.
33. A method for making the modified immune effector cell of claim 23 , the method comprising transfecting an immune effector cell with an expression vector encoding the chimeric polypeptide.
34. The method of claim 33 , wherein the vector is a Sleeping Beauty transposon.