# US 11,898,165 B2 · Engineered host cells and methods of use thereof

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- Number: US 11,898,165 B2
- Document ID: US11898165B2
- Status: Granted
- Kind: B2
- Application number: 15/759,007
- Filed: 2016-09-09
- Published: 2020-08-20
- Granted: 2024-02-13
- Earliest claimed: 2015-09-11
- Assignees: MiNK Therapeutics, Inc.
- Applicants: MiNk Therapeutics, Inc.
- Inventors: Marc Van Dijk; Volker Seibert; Robert Benjamin Stein
- Web page: https://patentgrep.com/patent/US11898165B2

## CPC

- C07K 14/70521: {CD28, CD152}
- 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)
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C12N 5/0636: {T lymphocytes}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C12N 15/86: Viral vectors
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/505: {involving T-cells}
- G01N 33/56972: {White blood cells}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/00: Fusion polypeptide
- C12N 2510/00: Genetically modified cells
- C12N 2740/10043: viral genome or elements thereof as genetic vector
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Provided herein are engineered host cells suitable for expression of functional T cell receptors (TCR) and methods of using these cells to identify TCRs that specifically bind to a desired peptide-major histocompatibility complex (MHC) complex.

## Claims

### Claim 1

An isolated cell derived from a mouse lymphocyte, the isolated cell expressing a chimeric CD8 on the cell surface, wherein the isolated cell comprises:
(a) a first polynucleotide encoding a chimeric CD8α chain comprising an extracellular region of human CD8α and transmembrane and cytoplasmic regions of mouse CD8α,
(b) a second polynucleotide encoding a chimeric CD8β chain comprising an extracellular region of human CD8β and transmembrane and cytoplasmic regions of mouse CD8β, wherein the isolated cell expresses CD3 but does not express endogenous TCR on the cell surface; and
(c) one or more reporter system for measurement of TCR signaling;
wherein the chimeric CD8α chain comprises the amino acid sequence of SEQ ID NO: 1, and/or the chimeric CD8β chain comprises the amino acid sequence of SEQ ID NO: 2.

### Claim 2 (depends on 1)

The isolated cell of claim 1, wherein:
(a) the one or more reporter systems provide a measurement of TCR signaling comprising one or more activities of the isolated cell selected from the group consisting of nuclear factor of activated T-cell (NFAT) signaling, IL-2 signaling, activator protein-I (API) signaling, NFKB/Rel signaling, and calcium flux; and
(b) the one or more reporter systems comprise a polynucleotide comprising a promoter region operably linked to a polynucleotide sequence encoding a detectable marker.

### Claim 3 (depends on 1)

The isolated cell of claim 1, wherein:
(a) the isolated cell is derived from a single cell and wherein when the cell is expanded to a plurality of cells of at least I 05 and transduced or transfected with separate polynucleotide vectors encoding a TCR α chain and a TCR β chain to express a recombinant TCR comprising the TCR α chain and the TCR β chain under conditions that maximize transduction or transfection efficiency, at least 20% of the plurality of cells express the recombinant TCR on the cell surface, optionally the cell has previously been subjected to one or more rounds of transduction or transfection;
(b) the mouse lymphocyte is a T cell, B cell, or an NK cell;
(c) the mouse lymphocyte is a T cell that is selected from the group consisting of a CD8+ cytotoxic T cell, a CD8+ regulatory T cell, a CD4+ cytotoxic T cell, a CD4+ helper T cell, and a CD4+ regulatory T cell; and/or
(d) the mouse lymphocyte is selected from the group consisting of a RAG1 or RAG2 knockout murine cell, an immature murine T cell, a mouse DO-11.10.7 58 α−β− (5831 /−) cell, and a mouse Bw5147 cell.

### Claim 4 (depends on 1)

The isolated cell of claim 1, wherein the cell further expresses a recombinant TCR on the cell surface, the recombinant TCR comprising a TCR a chain and a TCR β chain, wherein the TCR α chain and the TCR β chain each comprises a variable region and a non-variable region, each non-variable region comprising a constant region, a transmembrane region, and a cytoplasmic region, wherein for each chain, the variable region is linked to the constant region, the constant region is linked to the transmembrane region, and the transmembrane region is linked to the cytoplasmic region.

### Claim 5 (depends on 4)

The isolated cell of claim 4, wherein the recombinant TCR is a fusion molecule comprising a co-stimulatory signaling region.

### Claim 6 (depends on 1)

A cellular library comprising a plurality of the isolated cells of claim 1.

### Claim 7 (depends on 2)

The isolated cell of claim 2, wherein the promoter region is an IL-2 promoter or a minimal IL-2 promoter.

### Claim 8 (depends on 7)

The isolated cell of claim 7, wherein the IL-2 promoter comprises one or more transcriptional response elements comprising nuclear factor of activated T-cell (NFAT) response elements, activator protein-I (API) response elements, or NFKB/Rel response elements.

### Claim 9 (depends on 2)

The isolated cell of claim 2, wherein the IL-2 promoter comprises three NFAT binding sites.

### Claim 10 (depends on 2)

The isolated cell of claim 2, wherein the detectable marker is a fluorescent protein, a cell surface marker, or an antibiotic selection marker.

### Claim 11 (depends on 2)

The isolated cell of claim 2, wherein the detectable marker is enhanced green fluorescent protein (EGFP).

### Claim 12 (depends on 6)

The cellular library of claim 6, wherein the cellular library has a TCR diversity of at least 107, 108, or 109.