# US 2019/0169637 A1 · A method for high level and stable gene transfer in lymphocytes

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- Number: US 2019/0169637 A1
- Document ID: US20190169637A1
- Status: Published application
- Kind: A1
- Application number: 15/761,783
- Filed: 2016-09-22
- Published: 2019-06-06
- Earliest claimed: 2015-09-22
- Assignees: Julius-Maximilians-UniversitäT WüRzburg
- Applicants: Julius-Maximilians-Universität Würzburg
- Inventors: Michael HUDECEK; Zoltan IVICS
- Web page: https://patentgrep.com/patent/US20190169637A1

## CPC

- C12N 15/90: Stable introduction of foreign DNA into chromosome
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/31: Chimeric antigen receptors [CAR]
- A61K 40/4204: {Epidermal growth factor receptors [EGFR]}
- A61K 40/4211: {CD19 or B4}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- A61P 31/10: Antimycotics
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C07K 14/70503: {Immunoglobulin superfamily}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C07K 14/71: for growth factors; for growth regulators
- C07K 16/2803: {against the immunoglobulin superfamily}
- C12N 5/0636: {T lymphocytes}
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12Y 207/07: Nucleotidyltransferases (2.7.7)
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/02: containing a signal sequence
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12N 2800/50: Vectors for producing vectors
- C12N 2800/90: Vectors containing a transposable element
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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

The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the stable integration of transgenes into lymphocytes and other mammalian cells. The novel method is based on the use of an mRNA-encoded transposase (e.g. sleeping beauty transposase) in combination with a minicircle DNA-encoded transposable element. The novel method enables higher gene-transfer rates and is at the same time less toxic than the conventional approach, which is the use of plasmid DNA-encoded transposase in combination with a plasmid DNA-encoded transposable element. Applications of the invention include but are not limited to the stable integration of a transgene encoding an immune receptor (e.g. a T-cell receptor or synthetic chimeric antigen receptor) into human T lymphocytes, with the immune receptor conferring specificity for a molecule expressed by a tumor cell. The transposase mRNA and transposon minicircle DNA may be introduced into lymphocytes by methods including but not limited to electrotransfer such as electroporation and nucleofection.

## Claims

### Claim at position 1

1-110. (canceled)

### Claim 111

A method for obtaining a recombinant mammalian cell containing a stably integrated transposable element, the method comprising:
introducing a combination of a minicircle DNA encoding the transposable element and a transposase polypeptide or nucleic acid encoding a transposase into a mammalian cell, thereby obtaining the recombinant mammalian cell.

### Claim 112 (depends on 111)

The method according to claim 111, wherein the nucleic acid encoding the transposase is an mRNA encoding the transposase, a plasmid DNA encoding the transposase, a minicircle DNA encoding the transposase, or a linear DNA encoding the transposase and/or wherein the transposase or transposase polypeptide is SB100X.

### Claim 113 (depends on 111)

The method according to claim 111, wherein the transposable element contains the genetic information for the expression of a T-cell receptor or chimeric antigen receptor, and wherein the mammalian cell is a human T lymphocyte, and wherein the human T lymphocyte obtained by the method is a tumor-reactive human T lymphocyte suitable for use in the adoptive immunotherapy of cancer.

### Claim 114 (depends on 111)

The method according to claim 111, wherein the transposable element contains the genetic information for a chimeric antigen receptor, wherein the chimeric antigen receptor is specific for CD19, CD20, CD22, CD33, CD44v6, CD123, CD135, EpCAM, EGFR, an EGFR variant, GD2, ROR1, ROR2, CD269, CD319, CD38, or CD138.

### Claim 115 (depends on 111)

The method according to claim 111, wherein:
I) the minicircle DNA encoding the transposable element encodes an a/b or g/d T-cell receptor, a cytokine, a suicide gene, or a transduction marker; and/or
II) the method is an in vitro method; and/or
III) the nucleic acid encoding the transposase and the DNA minicircle encoding the transposable element are introduced into the cell by electrotransfer, such as electroporation, nucleofection; chemotransfer, calcium phosphate; or nanoparticles; and/or
IV) the transposase mediating transposition of the transposable element into the genome is Sleeping Beauty, PiggyBac, Frog Prince, Himarl, Passport, Minos, hAT, Tol1, Tol2, AciDs, PIF, Harbinger, Harbinger3-DR, and Hsmar1, or a derivative thereof having transposition activity; and/or
V) the combination of the minicircle DNA encoding the transposable element and the nucleic acid encoding the transposase are introduced together into the mammalian cell; and/or
VI) the nucleic acid encoding the transposase and the minicircle DNA encoding the transposable element are introduced into the mammalian cell in a molar ratio of 1:1, a molar ratio of 2:1 to 10:1, a molar ratio of 3:1 to 9:1, or a molar ratio of 4:1 to 8:1.

### Claim 116

A method for obtaining a recombinant mammalian cell containing a stably integrated transposable element, the method comprising:
introducing a combination of:
a DNA encoding a transposable element containing an expression cassette for a transgene and
a transposase polypeptide or a nucleic acid encoding a transposase into a mammalian cell,
thereby obtaining the recombinant mammalian cell,
wherein the DNA encoding the transposable element lacks an origin of replication and/or lacks an antibiotic resistance gene.

### Claim 117 (depends on 116)

The method according to claim 116, wherein the DNA encoding the transposable element is obtainable by deleting said origin of replication and/or said antibiotic resistance gene from a plasmid selected from the group consisting of:
pT;
pT2;
a plasmid having a DNA sequence which is at least 90% identical to the DNA sequence of pT;
a plasmid having a DNA sequence which is at least 90% identical to the DNA sequence of pT2; and
any other plasmid which is suitable as a donor plasmid for transposable elements.

### Claim 118

A method for obtaining a recombinant mammalian cell containing a stably integrated transposable element, the method comprising:
introducing a combination of:
a DNA encoding a transposable element containing an expression cassette for a transgene and
a transposase polypeptide or a nucleic acid encoding a transposase into a mammalian cell,
thereby obtaining the recombinant mammalian cell,
wherein the DNA encoding the transposable element is obtainable by shortening a plasmid by at least one base pair, and wherein the plasmid is selected from the group consisting of:
pT;
pT2;
a plasmid having a DNA sequence which is at least 90% identical to the DNA sequence of pT;
a plasmid having a DNA sequence which is at least 90% identical to the DNA sequence of pT2; and
any other plasmid which is suitable as a donor plasmid for transposable elements.

### Claim 119 (depends on 117)

The method according to claim 117, wherein:
I) the total length of said DNA encoding the transposable element is not more than 3.0 kb greater than the length of said expression cassette; and/or
II) the total length of said DNA encoding the transposable element is not more than 1.5 kb greater than the length of said expression cassette; and/or
III) the total length of said DNA encoding the transposable element is not more than 1.0 kb greater than the length of said expression cassette; and/or
IV) the DNA encoding the transposable element is a minicircle DNA; and/or
V) the transgene is a T-cell receptor or chimeric antigen receptor, and wherein the mammalian cell is a human T lymphocyte or the transgene is an a/b or g/d T-cell receptor, a cytokine, a suicide gene, or a transduction marker; and/or
VI) the method is an in vitro method; and/or
VII) the nucleic acid encoding the transposase is an mRNA encoding the transposase, a plasmid DNA encoding the transposase, a minicircle DNA encoding the transposase, or a linear DNA encoding the transposase; and/or
VIII) the method is a non-viral method; and/or
IX) said combination is introduced by introducing the DNA or minicircle DNA encoding the transposable element and the transposase polypeptide or nucleic acid encoding the transposase simultaneously.

### Claim 120 (depends on 116)

The method according to claim 116, wherein said combination is introduced by introducing
said transposase polypeptide or nucleic acid encoding the transposase
and said DNA or minicircle DNA encoding the transposable element sequentially; or
said combination is introduced by introducing
said DNA or minicircle DNA encoding the transposable element
and said transposase polypeptide or nucleic acid encoding the transposase sequentially.

### Claim 121 (depends on 116)

The method of claim 116, wherein:
I) the minicircle DNA encoding the transposable element is a linearized DNA or a circular DNA; and/or
II) the source of the transposase, or the nucleic acid encoding the transposase, is a minicircle DNA encoding the transposase, which is a linearized minicircle DNA or a circular minicircle DNA; and/or
III) the nucleic acid encoding the transposase and the DNA or minicircle DNA encoding the transposable element are introduced into the mammalian cell in a weight ratio of 1:1 or more, in a weight ratio of 2:1 to 10:1, in a weight ratio of 3:1 to 9:1, or in a weight ratio of 4:1 to 8:1.

### Claim 122 (depends on 111)

A recombinant mammalian cell obtainable by the method according to claim 111.

### Claim 123

A recombinant human T-cell containing at least one copy of a transposable element containing an expression cassette for a transgene.

### Claim 124 (depends on 123)

The recombinant cell of claim 123, wherein the copy number of the transposable element in said cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10; or wherein the copy number of the transposable element in said cell is at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10.

### Claim 125 (depends on 123)

The recombinant cell according to claim 123, wherein:
A) 0% to 5%; or
B) at least 5%; or
C) at least 10%; or
D) at least 15%; or
E) at least 20%; or
of the copies of the transposable element in the chromosomal genome of the recombinant cell are integrated in genomic chromosomal regions which satisfy all of the following criteria:
(i) not ultraconserved,
(ii) more than 300 kb away from miRNA genes,
(iii) more than 50 kb away from transcriptional start sites,
(iv) more than 300 kb away from genes involved in cancer, and
(v) outside transcription units.

### Claim 126 (depends on 123)

The recombinant cell according to claim 123, wherein at least 40% of the copies of the transposable element in the chromosomal genome of the recombinant cell are integrated in genomic chromosomal regions which satisfy the following criterion:
(v) outside transcription units.

### Claim 127 (depends on 123)

The recombinant cell claim 123, wherein one or all copies of the transposable element in the chromosomal genome of the recombinant cell are integrated in genomic chromosomal regions which satisfy at least:
A) any one; or
B) at least any two; or
C) at least any three; or
D) at least any four
of the following criteria:
(i) not ultraconserved,
(ii) more than 300 kb away from miRNA genes,
(iii) more than 50 kb away from transcriptional start sites,
(iv) more than 300 kb away from genes involved in cancer, and
(v) outside transcription units.

### Claim 128 (depends on 123)

The recombinant cell of claim 123, wherein the copy number of the transposable element in the chromosomal genome of the recombinant cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10; and/or wherein the copy number of transient copies of the transposable element in the recombinant cell is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10.

### Claim 129

A composition comprising a minicircle DNA encoding a transposable element and a transposase polypeptide or nucleic acid encoding the transposase.

### Claim 130 (depends on 133)

The composition according to claim 133, wherein the nucleic acid encoding the transposase is an mRNA encoding the transposase, a plasmid DNA encoding the transposase, a minicircle DNA encoding the transposase, or a linear DNA encoding the transposase; and/or wherein the transposable element contains the genetic information for the expression of a T-cell receptor or chimeric antigen receptor, and wherein the mammalian cell is a human T lymphocyte, and wherein the human T lymphocyte obtained by the method is a tumor-reactive human T lymphocyte suitable for use in the adoptive immunotherapy of cancer or contains the genetic information for a chimeric antigen receptor, wherein the chimeric antigen receptor is specific for CD19, CD20, CD22, CD33, CD44v6, CD123, CD135, EpCAM, EGFR, an EGFR variant, GD2, ROR1, ROR2, CD269, CD319, CD38, or CD138.