GHR-106 CHIMERIC ANTIGEN RECEPTOR CONSTRUCT AND METHODS OF MAKING AND USING SAME

• US 20190322754A1
• Filed: 12/22/2017
• Published: 10/24/2019
• Est. Priority Date: 01/01/2017
• Status: Abandoned Application

• Current Assignee: Gregory Chi-Yu Lee
• Original Assignee: Gregory Chi-Yu Lee
• Inventors: LEE, Gregory Chi-Yu
• Application Number:
  US16/474,936
• Publication Number:
  US 20190322754A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464402 Receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2869 against hormone receptors for antibodies against neuromediator receptors C07K16/286
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 2502/30 tumour cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 5/0638 Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]

Abstract

A chimeric antigen receptor (CAR) having an antigen binding domain capable of binding to extracellular domains of human GnRH receptor. The antigen binding domain can have a binding affinity and specificity similar to the GHR-106 antibody. Methods of making and using such CARs are provided. The CARs can be used to treat cancer.

Claims

1. A nucleotide vector capable of expressing a GHR-106 CAR.

2. A nucleotide vector as defined in claim 1, wherein the nucleotide vector encodes a polypeptide having from N-terminal to C-terminal:

an antigen binding domain capable of binding to an extracellular domain of human GnRH receptor;

a hinge domain;

a transmembrane domain; and

an intracellular T cell signaling domain.

3. A nucleotide vector as defined in claim 2, further encoding:

a signal peptide upstream of the N-terminal portion of the antigen binding domain.

4. A nucleotide vector as defined in claim 1, wherein the antigen binding domain comprises an scFv of GHR-106.

5. A nucleotide vector as defined in claim 2 wherein:

the antigen binding domain comprises V_H and V_L regions of a humanized GHR-106 monoclonal antibody joined by a linker;

the signal peptide comprises the interleukin 2 signaling sequence;

the hinge domain comprises the hinge domain of a CD8 molecule;

the transmembrane domain comprises the transmembrane domain of a CD8 molecule; and

/or

the intracellular T cell signaling domain comprises a CD3 zeta subunit domain.

6. A nucleotide vector as defined in claim 2, further comprising:

a costimulatory domain; and

/or

a cytokine positioned at the C-terminus of the intracellular T cell signaling domain and a self-cleavable peptide sequence interposing the intracellular T-cell signaling domain and the cytokine.

7. A nucleotide vector as defined in claim 6, wherein:

the costimulatory domain comprises a 4-1 BB costimulatory domain;

the cytokine comprises interleukin-7; and

/or

the self-cleavable peptide sequence comprises the peptide sequence of 2A.

8. A nucleotide vector as defined in claim 2 further comprising:

a promoter positioned to drive expression of the GHR-106 CAR;

a post-transcriptional regulatory element;

a 3′

LTR;

a 5′

LTR;

a transcription promoter;

a Gag sequence;

a Rev Response Element (RRE);

a gene encoding an envelope protein (Env);

a central polypurine tract;

an origin of replication; and

/or

an antibiotic resistance marker.

9. A nucleotide vector as defined in claim 8, wherein:

the promoter comprises EF-1 alpha promoter;

the post-transcriptional regulatory element comprises Woodchuck hepatitis virus post-transcriptional regulatory element;

the transcription promoter comprises a constitutive promoter, optionally Rous Sarcoma Virus (RSV) constitutive promoter; and

/or

the gene encoding the envelope protein (Env) comprises a gene encoding VSV-G envelope protein.

10. A nucleotide vector as defined in claim 5, wherein the V_H region of the humanized GHR-106 monoclonal antibody has the amino acid sequence of SEQ ID NO:

5, and/or wherein the V_L region of the humanized GHR-106 monoclonal antibody has the amino acid sequence of SEQ ID NO;

6.

11. A nucleotide vector as defined in claim 2, wherein the antigen-binding domain of the expressed protein binds to the extracellular domains of human GnRH receptor and with an affinity substantially equivalent to a humanized GHR-106 monoclonal antibody.

12. A nucleotide vector as defined in claim 1, having the general structure shown in FIG. 3

13. A nucleotide vector as defined in claim 1 that encodes an amino acid having the sequence of SEQ ID NO:

7.

16. A nucleotide vector as defined in claim 1, wherein the nucleotide vector comprises a lentiviral plasmid suitable for use as a transfer plasmid in a lentiviral vector system to transduce immune cells with a nucleotide sequence capable of expressing GHR-106 CAR.

20. A polypeptide encoded by a nucleotide vector as defined in claim 1, the polypeptide having the amino acid sequence of SEQ ID NO.7.

23. An immune cell comprising a nucleotide vector, polynucleotide molecule or isolated nucleic acid molecule as defined in claim 1.

14. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide having from N-terminal to C-terminal:

a signaling domain;

an antigen binding domain capable of binding to extracellular domains of the human GnRH receptor;

a transmembrane domain;

a CD3-zeta signaling domain; and

a cytokine domain separated from the CD3-zeta signaling domain by a self-cleavable peptide.

15. (canceled)

17. A GHR-106 CAR comprising from N-terminal to C-terminal:

an antigen binding domain capable of binding to an extracellular domain of human GnRH receptor;

a transmembrane domain; and

an intracellular T cell signaling domain.

18. A GHR-106 CAR as defined in claim 17, further comprising

a signal peptide positioned on the N-terminal side of the antigen binding domain;

a hinge domain; and

an intracellular costimulatory domain.

19. A GHR-106 CAR as defined in claim 17, wherein:

the antigen binding domain comprises V_H and V_L regions of a humanized GHR-106 monoclonal antibody joined by a linker;

the signal peptide comprises the interleukin 2 signaling sequence;

the hinge domain comprises the hinge domain of a CD8 molecule;

the transmembrane domain comprises the transmembrane domain of a CD8 molecule;

the intracellular T cell signaling domain comprises a CD3 zeta subunit domain; and

/or

the intracellular costimulatory domain comprises a 4-1BB costimulatory domain.

21. (canceled)

22. (canceled)

24-40. -40. (canceled)