# US 11,298,378 B2 · Materials and methods for engineering cells and uses thereof in immuno-oncology

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- Number: US 11,298,378 B2
- Document ID: US11298378B2
- Status: Granted
- Kind: B2
- Application number: 17/228,542
- Filed: 2021-04-12
- Published: 2021-08-05
- Granted: 2022-04-12
- Earliest claimed: 2017-05-12
- Assignees: Crispr Therapeutics AG
- Applicants: CRISPR Therapeutics AG
- Inventors: Jonathan Alexander Terrett; Demetrios Kalaitzidis; Lawrence Klein
- Web page: https://patentgrep.com/patent/US11298378B2

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- 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 40/421: {Immunoglobulin superfamily}
- A61K 40/4211: {CD19 or B4}
- A61K 40/4213: {CD74, Ii, MHC class II invariant chain or MHC class II gamma chain}
- A61K 40/4215: {Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30}
- A61K 40/4232: {Tumor necrosis factors [TNF] or CD70}
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61K 48/0066: {Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid}
- A61P 35/00: Antineoplastic agents
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C07K 14/70517: {CD8}
- C07K 14/70521: {CD28, CD152}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- C07K 14/70578: {NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 (NGF-receptor C07K14/71, TNF-receptor C07K14/7151)}
- C12N 5/0634: {Cells from the blood or the immune system}
- C12N 5/0636: {T lymphocytes}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/86: Viral vectors
- 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)
- A61K 40/50: characterised by the use of allogeneic cells
- 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
- A61K 2239/56: Kidney
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/03: containing a transmembrane segment
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 15/907: {in mammalian cells}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)

## Abstract

Materials and methods for producing genome-edited cells engineered to express a chimeric antigen receptor (CAR) construct on the cell surface, and materials and methods for genome editing to modulate the expression, function, or activity of one or more immuno-oncology related genes in a cell, and materials and methods for treating a patient using the genome-edited engineered cells.

## Claims

### Claim 1

A method for treating a BCMA+ cancer, comprising administering to a subject in need thereof an effective amount of a population of engineered human T cells,
wherein the engineered human T cells express a chimeric antigen receptor (CAR) that binds B-cell maturation antigen (BCMA),
wherein the CAR comprises an ectodomain comprising an anti-BCMA single-chain variable fragment (scFv), and
wherein the anti-BCMA scFv comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1524, or 1589, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1526, 1527, or 1590.

### Claim 2 (depends on 1)

The method of claim 1, wherein the anti-BCMA scFv comprises:
(a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1524 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:1526;
(b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1524 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:1527; or
(c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1589 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:1590.

### Claim 3 (depends on 1)

The method of claim 1, wherein the anti-BCMA scFv comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1589 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:1590.

### Claim 4 (depends on 1)

The method of claim 1, wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 1503 or 1509.

### Claim 5 (depends on 4)

The method of claim 4, wherein the anti-BCMA scFv comprises the amino acid sequence of SEQ ID NO: 1509.

### Claim 6 (depends on 1)

The method of claim 1, wherein the CAR further comprises a CD8 transmembrane domain, and an endodomain, and wherein the endodomain comprises a: i) 4-1BB or CD28 co-stimulatory domain and ii) a CD3z co-stimulatory domain.

### Claim 7 (depends on 6)

The method of claim 6, wherein the endodomain comprises a 4-1BB co-stimulatory domain.

### Claim 8 (depends on 6)

The method of claim 6, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 1453, 1454, 1460, or 1461.

### Claim 9 (depends on 8)

The method of claim 8, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 1461.

### Claim 10 (depends on 1)

The method of claim 1, wherein the engineered human T cells comprise:
(a) a disrupted TRAC gene;
(b) a nucleic acid comprising a nucleotide sequence encoding the CAR; wherein the nucleic acid is inserted in the disrupted TRAC gene; and
(c) a disrupted beta-2-microglobulin (B2M) gene.

### Claim 11 (depends on 10)

The method of claim 10, wherein the nucleotide sequence encoding the CAR comprises SEQ ID NO: 1427, 1428, 1434, or 1435.

### Claim 12 (depends on 11)

The method of claim 11, wherein the nucleotide sequence encoding the CAR comprises SEQ ID NO: 1435.

### Claim 13 (depends on 10)

The method of claim 10, wherein the disrupted TRAC gene comprises a deletion of SEQ ID NO:76.

### Claim 14 (depends on 13)

The method of claim 13, wherein the nucleic acid of (b) comprises the nucleotide sequence of SEQ ID NO: 1409.

### Claim 15 (depends on 14)

The method of claim 14, wherein the disrupted B2M gene comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1560; SEQ ID NO: 1561; SEQ ID NO: 1562; SEQ ID NO: 1563; SEQ ID NO: 1564; and SEQ ID NO: 1565.

### Claim 16 (depends on 10)

The method of claim 10, wherein the nucleic acid of (b) comprises the nucleotide sequence of SEQ ID NO: 1401, 1402, 1408, or 1409.

### Claim 17 (depends on 10)

The method of claim 10, wherein the disrupted B2M gene comprises an insertion of at least one nucleotide base pair, a deletion of at least one nucleotide base pair, or a combination thereof.

### Claim 18 (depends on 10)

The method of claim 10, wherein at least 90% of the engineered human T cells in the population do not express a detectable level of TCR surface protein.

### Claim 19 (depends on 10)

The method of claim 10, wherein at least 50% of the engineered human T cells in the population express a detectable level of the CAR.

### Claim 20 (depends on 10)

The method of claim 10, wherein at least 70% of the engineered human T cells in the population do not express a detectable level of TCR surface protein, wherein at least 50% of the engineered human T cells in the population do not express a detectable level of B2M surface protein, and wherein at least 50% of the engineered human T cells in the population express a detectable level of the CAR.

### Claim 21 (depends on 20)

The method of claim 20, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 1461.

### Claim 22 (depends on 10)

The method of claim 10, wherein the population of the engineered human T cells is allogenic to the subject.

### Claim 23 (depends on 10)

The method of claim 10, wherein the population of the engineered human T cells is from one or more healthy donors.

### Claim 24 (depends on 10)

The method of claim 10, wherein the population of the engineered human T cells reduces tumor volume in the subject.

### Claim 25 (depends on 10)

The method of claim 10, wherein the population of the engineered human T cells increases survival rate in the subject.

### Claim 26 (depends on 10)

The method of claim 10, wherein the subject is a human patient having a BCMA+ cancer.

### Claim 27 (depends on 26)

The method of claim 26, wherein the human patient has multiple myeloma, or plasmacytoma.

### Claim 28 (depends on 1)

The method of claim 1, wherein the population of the engineered human T cells is suspended in a pharmaceutically acceptable carrier.

### Claim 29 (depends on 1)

The method of claim 1, wherein the population of the engineered human T cells is administered to the subject via an intravenous route.