# US 11,077,176 B2 · Methods for inducing selective apoptosis

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- Number: US 11,077,176 B2
- Document ID: US11077176B2
- Status: Granted
- Kind: B2
- Application number: 15/886,309
- Filed: 2018-02-01
- Published: 2018-08-30
- Granted: 2021-08-03
- Earliest claimed: 2010-05-21
- Assignees: Baylor College of Medicine
- Applicants: Baylor College of Medicine
- Inventors: Malcolm K. Brenner
- Web page: https://patentgrep.com/patent/US11077176B2

## CPC

- A61K 38/52: Isomerases (5)
- 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)
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61K 38/4873: {Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H}
- A61K 39/001: {Preparations to induce tolerance to non-self, e.g. prior to transplantation}
- A61K 40/10: characterised by the cell type used
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/22: Immunosuppressive or immunotolerising
- A61K 40/418: {Antigens related to induction of tolerance to non-self}
- A61K 40/4211: {CD19 or B4}
- A61K 40/46: Viral antigens
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 35/00: Antineoplastic agents
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 5/0636: {T lymphocytes}
- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- C07K 2319/00: Fusion polypeptide
- C12N 2501/48: Regulators of apoptosis
- C12N 2510/00: Genetically modified cells
- C12Y 304/22062: Caspase-9 (3.4.22.62)
- C12Y 502/01008: Peptidylprolyl isomerase (5.2.1.8), i.e. cyclophilin
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Provided herein are methods for cell therapy by modifying transfused cells to express an inducible caspase 9 protein, so that the cells may be selectively killed if the patient experiences dangerous side effects. Provided also within relates in part to methods for preventing or treating Graft versus Host Disease by modifying T cells before administration to a patient, so that they may be selectively killed if GvHD develops in the patient.

## Claims

### Claim 1

A method of treating a patient with therapeutic cells, comprising administering a multimeric ligand to a human patient to whom therapeutic cells have been administered, wherein:
the therapeutic cells comprise modified T cells;
the therapeutic cells have been transfected or transduced with a nucleic acid comprising (i) a promoter region, and (ii) a polynucleotide that encodes a chimeric protein comprising a multimeric ligand binding region and a caspase 9 polypeptide;
the promoter region is operatively linked to the polynucleotide;
the multimeric ligand binding region binds to a multimeric ligand;
the multimeric ligand binding region comprises a modified FKBP12 polypeptide;
the modified FKBP12 polypeptide binds with higher affinity to the multimeric ligand than the wild type FKBP12 polypeptide;
the modified FKBP12 polypeptide comprises an amino acid substitution at position 36;
activated therapeutic cells are depleted at a rate greater than 90% depletion in one day following administration of the multimeric ligand; and
the activated therapeutic cells are CD3-positive therapeutic cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the multimeric ligand is AP1903.

### Claim 3 (depends on 1)

The method of claim 1, wherein the multimeric ligand is AP20187.

### Claim 4 (depends on 1)

The method of claim 1, wherein the therapeutic cells comprise allogeneic T cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the therapeutic cells are non-allodepleted.

### Claim 6 (depends on 1)

The method of claim 1, wherein the caspase-9 polypeptide is a truncated caspase-9 polypeptide.

### Claim 7 (depends on 1)

The method of claim 1, wherein the modified FKBP12 polypeptide binds with higher affinity to AP1903 than the wild type FKBP12 polypeptide.

### Claim 8 (depends on 1)

The method of claim 1, wherein the FKBP12 polypeptide comprises a valine at position 36.

### Claim 9 (depends on 1)

The method of claim 1, further comprising administering a haploidentical stem cell transplant to the patient prior to administering the therapeutic cells.

### Claim 10 (depends on 1)

The method of claim 1, wherein the chimeric protein further comprises a marker polypeptide.

### Claim 11 (depends on 1)

The method of claim 1, wherein the method comprises the activated therapeutic cells being depleted at a rate greater than 90% depletion within one hour following administration of the multimeric ligand.

### Claim 12 (depends on 1)

The method of claim 1, wherein the FKBP12 polypeptide is encoded by a polynucleotide comprising one or more wobbled codons.

### Claim 13 (depends on 4)

The method of claim 4, wherein the allogeneic T cells are not in an active state when administered to the patient.

### Claim 14 (depends on 4)

The method of claim 4, wherein the allogeneic T cells are over 90% pure.

### Claim 15 (depends on 1)

The method of claim 1, wherein the T cells comprise cytotoxic T cells.

### Claim 16 (depends on 1)

The method of claim 1, wherein the T cells comprise T cells reactive to tumor cells in the patient.

### Claim 17 (depends on 1)

The method of claim 1, wherein the T cells comprise tumor-associated antigen specific T cells.

### Claim 18 (depends on 1)

The method of claim 1, wherein the T cells comprise virus-specific T cells.

### Claim 19

A method of treating a patient with therapeutic cells, comprising administering a multimeric ligand to a human patient to whom therapeutic cells have been administered, wherein:
the therapeutic cells comprise modified T cells;
the therapeutic cells have been transfected or transduced with a nucleic acid comprising (i) a promoter region, and (ii) a polynucleotide that encodes a chimeric protein comprising a multimeric ligand binding region and a caspase 9 polypeptide;
the promoter region is operatively linked to the polynucleotide;
the multimeric ligand binding region binds to a multimeric ligand;
the multimeric ligand is AP20187 or AP1903;
the multimeric ligand binding region comprises a modified FKBP12 polypeptide;
the modified FKBP12 polypeptide binds with higher affinity to the multimeric ligand than the wild type FKBP12 polypeptide;
activated therapeutic cells are depleted at a rate greater than 90% depletion in one day following administration of the multimeric ligand: and
the activated therapeutic cells are CD3-positive therapeutic cells.

### Claim 20 (depends on 19)

The method of claim 19, wherein the multimeric ligand is AP1903.

### Claim 21 (depends on 19)

The method of claim 19, wherein the multimeric ligand is AP20187.

### Claim 22 (depends on 19)

The method of claim 19, wherein the therapeutic cells comprise allogeneic T cells.

### Claim 23 (depends on 19)

The method of claim 19, wherein the therapeutic cells are non-allodepleted.

### Claim 24 (depends on 19)

The method of claim 19, wherein the caspase-9 polypeptide is a truncated caspase-9 polypeptide.

### Claim 25 (depends on 19)

The method of claim 19, wherein the modified FKBP12 polypeptide binds with higher affinity to AP1903 than the wild type FKBP12 polypeptide.

### Claim 26 (depends on 19)

The method of claim 19, wherein the FKBP12 polypeptide comprises a valine at position 36.

### Claim 27 (depends on 19)

The method of claim 19, further comprising administering a haploidentical stem cell transplant to the patient prior to administering the therapeutic cells.

### Claim 28 (depends on 19)

The method of claim 19, wherein the chimeric protein further comprises a marker polypeptide.

### Claim 29 (depends on 19)

The method of claim 19, wherein the method comprises the activated therapeutic cells being depleted at a rate greater than 90% depletion within one hour following administration of the multimeric ligand.

### Claim 30 (depends on 19)

The method of claim 19, wherein the FKBP12 polypeptide is encoded by a polynucleotide comprising one or more wobbled codons.

### Claim 31 (depends on 22)

The method of claim 22, wherein the allogeneic T cells are not in an active state when administered to the patient.

### Claim 32 (depends on 22)

The method of claim 22, wherein the allogeneic T cells are over 90% pure.

### Claim 33 (depends on 19)

The method of claim 19, wherein the T cells comprise cytotoxic T cells.

### Claim 34 (depends on 19)

The method of claim 19, wherein the T cells comprise T cells reactive to tumor cells in the patient.

### Claim 35 (depends on 19)

The method of claim 19, wherein the T cells comprise tumor-associated antigen specific T cells.

### Claim 36 (depends on 19)

The method of claim 19, wherein the T cells comprise virus-specific T cells.