CHIMERIC ANTIGEN RECEPTORS WITH AN OPTIMIZED HINGE REGION

• US 20130280285A1
• Filed: 09/06/2011
• Published: 10/24/2013
• Est. Priority Date: 09/08/2010
• Status: Active Grant

• Current Assignee: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus
• Original Assignee: Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus
• Inventors: Knopp, Christiane, Wels, Winfried, Schnfeld, Kurt
• Granted Patent: US 9,212,229 B2
• Time in Patent Office: Days
• US Class Current: 424/185.1
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 CD8
• CPC Class Codes: C07K 14/70521 CD28, CD152
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/626 Diabody or triabody
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

The present invention relates to multi-functional proteins which comprise (i) a signal peptide, (ii) a target specific recognition domain, (iii) a linker region, connecting domain (ii) and domain (iv) which comprises a specific modified hinge region of the human CD8 alpha-chain, and (iv) an effector domain. The present invention furthermore relates to nucleic acids encoding the proteins, expression constructs for expressing the protein in a host cell and host cells. The proteins of the invention are chimeric antigen receptors with an optimized linker or hinge region that are suitable for generating target-specific effector cells, for use as a medicament, in particular in the treatment of cancer and in adoptive, target-cell specific immunotherapy.

Claims

1. A protein comprising

(i) a signal peptide;

(ii) a target specific recognition domain;

(iii) a linker region, connecting domain (ii) and domain (iv),

wherein the linker region does not contain cysteine residue(s) and is selected from the following;

the amino acid sequence of SEQ ID NO. 2,

an amino acid sequence with at least 95% sequence identity to SEQ ID NO. 2 under the proviso that amino acid residue 48 is not a cysteine and is a serine, and

an amino acid sequence that differs in one, two or three amino acid residues from the amino acid sequence of SEQ ID NO. 2 under the proviso that amino acid residue 48 is not a cysteine and is a serine; and

(iv) an effector domain comprising a transmembrane region and one or more intracellular signaling domains,

wherein the effector domain (iv) comprises, or is, a fusion of a fragment of a human costimulatory receptor fused to a fragment of a zeta-chain of a human CD3 complex of a T-cell receptor.

2. The protein of claim 1, wherein the target is a cell or a virus and wherein the target specific recognition domain (ii) binds an antigen, receptor, peptide ligand or protein ligand of the target.

3. The protein of claim 1, wherein the target specific recognition domain (ii) comprises

an antigen binding domain derived from an antibody against an antigen of the target, or

a peptide that binds an antigen of the target, or

a peptide or protein that binds an antibody that binds an antigen of the target, or

a peptide or protein ligand that binds a receptor on the target, or

a domain derived from a receptor that binds a peptide or protein ligand on the target.

4. The protein according to claim 1, wherein the antigen of the target is a tumor-associated surface antigen, a lineage-specific or tissue-specific surface antigen or a virus-specific surface antigen.

5. The protein according to claim 1, wherein the antigen binding domain of domain (ii) is derived from an antibody or an antibody fragment, such as a single chain Fv (scFv) fragment, Fab fragment, a diabody, a variable domain of the antibody heavy chain or antibody light chain.

6. The protein according to claim 1, wherein the effector domain (iv) comprises or is

a fusion of a fragment of the human costimulatory CD28 receptor fused to a fragment of the zeta-chain of the human CD3 complex of the T-cell receptor.

7. The protein according to claim 1, comprising the amino acid sequence of SEQ ID NO. 7,

or an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 7 under the proviso that amino acid residue no. 308 is not a cysteine and is a serine.

8. A nucleic acid encoding the protein according to claim 1.

9. The nucleic acid of claim 8, comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO. 2 or comprising a nucleic acid sequence of SEQ ID NO. 8, or their complementary sequences or sequences that have at least 95% sequence identity.

10. The nucleic acid of claim 8, comprising the nucleic acid encoding the amino acid sequence of SEQ ID NO. 5 or comprising the nucleic acid sequence of SEQ ID NO. 10, or their complementary sequences or sequences that have at least 95% sequence identity.

11. The nucleic acid of claim 8, comprising the nucleic acid encoding the amino acid sequence of SEQ ID NO. 7 or the nucleic acid sequence of SEQ ID NO. 12, or their complementary sequences or sequences that have at least 95% sequence identity.

12. An expression construct for expressing, in a cell, a protein comprising

(i) a signal peptide;

(ii) a target specific recognition domain;

(iii) a linker region, connecting domain (ii) and domain (iv),

wherein the linker region does not contain cysteine residue(s) and is selected from the following;

the amino acid sequence of SEQ ID NO. 2,

an amino acid sequence with at least 95% sequence identity to SEQ ID NO. 2 under the proviso that amino acid residue 48 is not a cysteine and is a serine, and

an amino acid sequence that differs in one, two or three amino acid residues from the amino acid sequence of SEQ ID NO. 2 under the proviso that amino acid residue 48 is not a cysteine and is a serine; and

(v) an effector domain comprising a transmembrane region and one or more intracellular signaling domains,

wherein the effector domain (iv) comprises, or is, a fusion of a fragment of a human costimulatory receptor fused to a fragment of a zeta-chain of a human CD3 complex of a T-cell receptor, and

wherein said construct comprises a nucleic acid of claim 8.

13. A host cell expressing a protein of claim 1 or comprising a nucleic acid encoding the protein of claim 1,

wherein the host cell is selected from effector cells of the immune system, and wherein the effector cells of the immune system are natural killer (NK) cells, natural killer T (NKT) cells, or a lymphocyte preparation containing NK cells and NKT cells mast cells.

14. A method for generating target-specific effector cells wherein said method comprises the use of a protein of claim 1 or a nucleic acid encoding a protein of claim 1.

15. A pharmaceutical composition comprising the protein of claim 1 or a nucleic acid encoding the protein of claim 1 and a pharmaceutical carrier.

16. A method for the treatment of cancer or for use in adoptive target-cell specific immunotherapy, wherein said method comprises administering, to a subject in need of such cancer treatment or adoptive target-cell specific immunotherapy, the protein of claim 1 or a nucleic acid encoding the protein of claim 7.