SORTASE SYNTHESIZED CHIMERIC ANTIGEN RECEPTORS

• US 20170211055A1
• Filed: 07/21/2015
• Published: 07/27/2017
• Est. Priority Date: 07/21/2014
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Guimaraes, Carla, Brogdon, Jennifer, Hastewell, John, Loew, Andreas
• Granted Patent: US 11,542,488 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: C07K 2319/70 containing domain for protein-protein interaction
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea

Abstract

Sortase molecules and methods described herein allow for the construction of a CAR or CAR member, e.g., in situ, on a CARX, e.g., CART, cell. For example, sortase mediated transfer of an antigen binding domain, e.g., a scFv, onto a CAR member having a sortase acceptor motif in place of an antigen binding domain can provide for a complete CAR member on a cell wherein the cell does not comprise nucleic acid that encodes the complete CAR member.

Claims

1. A sortase acceptor member comprising:

(i) a sortase acceptor motif;

(ii) a transmembrane domain; and

optionally

(iii) an intracellular signaling domain or (iv) a switch domain.

2. The sortase acceptor member of claim 1, wherein the components, in order of N terminal to C terminal, is (i), (ii), and (iii) or (iv).

3. The sortase acceptor member of claim 1, comprising an intracellular signaling domain.

4. The sortase acceptor member of claim 1, comprising a switch domain.

5. The sortase acceptor member of claim 1, comprising a moiety that accepts transfer of a moiety by a sortase.

6. The sortase acceptor member of claim 3, wherein the intracellular signaling domain comprises a primary intracellular signaling domain, which is optionally selected from the list in Table 8.

7. The sortase acceptor member of claim 3, wherein intracellular signaling domain comprises a costimulatory signaling domain which is optionally selected from the list in Table 9.

8. The sortase acceptor member of claim 3, comprising a second intracellular signaling domain.

9. A nucleic acid or a vector encoding a sortase acceptor member of claim 1.

10. A cell, or preparation of cells, comprising a nucleic acid encoding a sortase acceptor member of claim 1.

11. The cell of claim 10, wherein the cell is a T cell or an NK cell.

12. A CAR member comprising, in order of N terminal to C terminal:

(i) an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain;

(ii) a sortase transfer signature;

(iii) a transmembrane domain; and

optionally,

(iv) an intracellular signaling domain or (iv) a switch domain.

13. The CAR member of claim 12, wherein the sortase transfer signature comprises an amino acid residue, which is optionally an X1 amino acid residue, from a sortase recognition motif from any of Tables 1-6.

14. The CAR member of claim 12, wherein the sortase transfer signature comprises an amino acid residue from the sequence LPXT (SEQ ID NO:

30), wherein X is any amino acid.

18. A CARX cell comprising the CAR member of claim 12.

15. A method of providing a cell comprising a CAR member comprising an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain, a transmembrane domain, and an intracellular signaling domain or a switch domain, comprising:

a) providing a precursor cell comprising a sortase acceptor member comprising;

(i) a sortase acceptor motif;

(ii) a transmembrane domain; and

(iii) an intracellular signaling domain or (iv) a switch domain

b) contacting the precursor cell with

(i) a sortase molecule and a polypeptide comprising an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain and a sortase recognition motif;

or

(ii) a complex comprising a polypeptide comprising an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain, a cleaved sortase recognition site, and a sortase;

under conditions sufficient to allow transfer of the polypeptide comprising an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain and a cleaved sortase recognition motif to the sortase acceptor motif on the sortase acceptor member,

thereby providing the cell.

16. The method of claim 15, wherein the sortase is a calcium independent sortase, a sortase A, or a Staphylococcus aureus sortase.

17. A polypeptide comprising an antigen binding domain, an extracellular domain (ECD) of an inhibitory molecule, or a costimulatory ECD domain, optionally attached as a chimeric polypeptide, to a sortase recognition motif.