# US 2021/0340530 A1 · Compositions and Methods for Immunotherapy

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- Number: US 2021/0340530 A1
- Document ID: US20210340530A1
- Status: Published application
- Kind: A1
- Application number: 17/231,556
- Filed: 2021-04-15
- Published: 2021-11-04
- Earliest claimed: 2018-10-16
- Assignees: Intellia Therapeutics, Inc.
- Applicants: Intellia Therapeutics, Inc.
- Inventors: Amy Melissa Becker; Surbhi Goel; Sarah Beth Hesse; Troy Aaron Luster; Birgit Schultes; Stephanie A. Yazinski; Pooja Vinay
- Web page: https://patentgrep.com/patent/US20210340530A1

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/7051: {T-cell receptor (TcR)-CD3 complex}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- 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 2320/30: Special therapeutic applications
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- 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

Compositions and methods for editing, e.g., altering a DNA sequence, within the TRBC1, TRBC2 and/or TRAC genes are provided. Compositions and methods for immunotherapy are provided, for example.

## Claims

### Claim 1

A method of altering a DNA sequence within the TRBC1 and/or TRBC2 gene, or reducing the expression of the TRBC1 and/or TRBC2 gene, comprising delivering a composition to a cell, wherein the composition comprises:
(a) a guide RNA comprising a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89;
iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and
v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or
(b) a nucleic acid encoding a guide RNA of (a); and
(c) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 2

A method of altering a DNA sequence, comprising delivering a composition to a cell, wherein the composition comprises:
(a) a guide RNA comprising a sequence chosen from:
i. a sequence that comprises 15 consecutive nucleotides ±10 nucleotides of a genomic coordinate listed in any of Tables 1 and/or 3 for SEQ ID NOs: 1-89 and 179-184;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (i);
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from (i);
(b) a nucleic acid encoding a guide RNA of (a); and
(c) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 3

(canceled)

### Claim 4

A method of immunotherapy comprising administering a composition to a subject, an autologous cell thereof, and/or an allogeneic cell, wherein the composition comprises:
a. a guide RNA comprising a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89;
iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and
v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or
b. a nucleic acid encoding a guide RNA of (a); and
c. an RNA-guided DNA binding agent or nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 5

A method of altering a DNA sequence within the TRAC gene, or reducing the expression of the TRAC gene, comprising delivering a composition to a cell, wherein the composition comprises:
a. a guide RNA comprising a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iv. a guide sequence comprising any one of SEQ ID NOs: 90-113, 185, and 213-218; and
v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or
b. a nucleic acid encoding a guide RNA of (a); and
c. an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 6

A method of altering a DNA sequence, comprising delivering a composition to a cell, wherein the composition comprises:
(a) a guide RNA comprising a sequence chosen from:
i. a sequence that comprises 15 consecutive nucleotides ±10 nucleotides of a genomic coordinate listed in any of Tables 2 and/or 3 for SEQ ID NOs: 90-218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (i);
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from (i);
(b) a nucleic acid encoding a guide RNA of (a); and
(c) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 7

(canceled)

### Claim 8

A method of immunotherapy comprising administering a composition to a subject, an autologous cell thereof, and/or an allogeneic cell, wherein the composition comprises:
a. a guide RNA comprising a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iv. a guide sequence comprising any one of SEQ ID NOs: 90-113, 185, and 213-218; and
v. a guide sequence comprising any one of SEQ ID NOs: 90-95; or
b. a nucleic acid encoding a guide RNA of (a.); and
c. an RNA-guided DNA binding agent or nucleic acid encoding an RNA-guided DNA binding agent.

### Claim 9

A method of altering a DNA sequence within the TRBC1, TRBC2 and/or TRAC genes, or reducing the expression of the TRBC1, TRBC2 and/or TRAC genes, comprising delivering to a cell a first guide RNA, a second guide RNA and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the first guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89;
iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and
v. a guide sequence comprising any one of SEQ ID NOs: 1-6, and
wherein the second guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iv. a guide sequence comprising any one of SEQ ID NOs: 90-113, 185, and 213-218; and
v. a guide sequence comprising any one of SEQ ID NOs: 90-95.

### Claim 10

(canceled)

### Claim 11

A method of immunotherapy comprising administering a composition to a subject, an autologous cell thereof, or an allogeneic cell, wherein the composition comprises: a first guide RNA, a second guide RNA, and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the first guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89;
iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; and
v. a guide sequence comprising any one of SEQ ID NOs: 1-6, and
wherein the second guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218;
iv. a guide sequence comprising any one of SEQ ID NOs: 90-113, 185, and 213-218; and
v. a guide sequence comprising any one of SEQ ID NOs: 90-95.

### Claim 12

A method of expressing a heterologous immunological receptor via in locus insertion at the TRAC locus, comprising delivering to a cell a first guide RNA, a second guide RNA and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the first guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 1-89;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89;
iv. a guide sequence comprising any one of SEQ ID NOs: 1, 2, 3, 5, 6; and
v. a guide sequence comprising any one of SEQ ID NOs: 2, 3, 5, 6, and
wherein the second guide RNA comprises a sequence chosen from:
i. a guide sequence selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, 218;
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218;
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218;
iv. a guide sequence comprising any one of SEQ ID NOs: 90, 95, 97, 185, and 214;
v. a guide sequence comprising any one of SEQ ID NOs: 90, 95, and 185; and
vi. a guide sequence comprising SEQ ID NO: 90 or 214.

### Claim at position 13

13-14. (canceled)

### Claim 15 (depends on 9)

The method of claim 9, wherein the first guide RNA comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 5, 6, and the second guide RNA comprises the sequence of SEQ ID NO. 90.

### Claim 16 (depends on 9)

The method of claim 9, wherein the first guide RNA comprises the sequence of any one of SEQ ID NOs: 1, 2, 3, 5, 6, and the second guide RNA comprises the sequence of any one of SEQ ID NOs: 95, 97, 98, 214, and 218.

### Claim 17 (depends on 9)

The method of claim 9, wherein the first guide RNA, the second guide RNA and the RNA-guided DNA binding agent or the nucleic acid encoding an RNA-guided DNA binding agent are administered at substantially the same time.

### Claim at position 17

18-20. (canceled)

### Claim 21 (depends on 5)

The method of claim 5, further comprising introducing a nucleic acid sequence encoding a polypeptide of interest, wherein:
a. the one or more polypeptides of interest comprise a receptor;
b. the one or more polypeptides of interest comprise an immunological receptor;
c. the one or more polypeptides of interest comprise a T-cell receptor, further optionally wherein the T-cell receptor recognizes a cancer antigen;
d. the one or more polypeptides of interest comprise a WT1-specific T-cell receptor, wherein the T-cell receptor recognizes WT1 or a fragment thereof;
e. the one or more polypeptides of interest comprise a chimeric antigen receptor, further wherein the chimeric antigen receptor recognizes a cancer antigen; or
f. the one or more polypeptides of interest comprise a WT1-specific chimeric antigen receptor, wherein the chimeric antigen receptor recognizes WT1 or a fragment thereof.

### Claim 22 (depends on 5)

The method of claim 5, further comprising:
a. introducing a TCR α chain and a TCR β chain;
b. introducing one or more nucleic acid sequences that encode a TCR α chain and a TCR β chain;
c. introducing a WT1-specific TCR α chain and β chain;
d. introducing one or more nucleic acid sequences that encode a WT1-specific TCR α chain and β chain;
e. introducing a first TCR sequence selected from: (i) SEQ ID NO. 501 or 504; (ii) an amino acid sequence that is at least 95% or 90% identical to SEQ ID NO. 501 or 504; and (iii) a contiguous subsequence of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250 amino acids of SEQ ID NO. 501 or 504,
and
introducing a second TCR sequence selected from: (i) SEQ ID NO. 502 or 505; (ii) an amino acid sequence that is at least 95% or 90% identical to SEQ ID NO. 502 or 505; and (iii) a contiguous subsequence of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids of SEQ ID NO. 502 or 505,
f. introducing a first TCR sequence selected from: (i) SEQ ID NO.501 or 513; (ii) an amino acid sequence that is at least 95% or 90% identical to SEQ ID NO.510 or 513; and (iii) a contiguous subsequence of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 250 amino acids of SEQ ID NO.510 or 513,
and
introducing a second TCR sequence selected from: (i) SEQ ID NO.511 or 514; (ii) an amino acid sequence that is at least 95% or 90% identical to SEQ ID NO.511 or 514; and (iii) a contiguous subsequence of at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 amino acids of SEQ ID NO.511 or 514,
g. introducing a nucleic acid sequence comprising a sequence that encodes a first TCR sequence of (e) or (f);
h. introducing a nucleic acid sequence comprising a sequence that encodes a second TCR sequence of (e) or (f);
i. introducing a nucleic acid sequence comprising the nucleic acid sequence of (g) and (h);
j. introducing a polypeptide selected from SEQ ID NOs: 500, 503, 506, 509, 512, 515, 518, or 521 or an amino acid sequence that is at least 95% or 90% identical thereto, by introducing a nucleic acid sequence encoding the same;
k. introducing a TCR α chain and a TCR β chain polypeptides selected from (i)-(vii) below, or an amino acid sequence that is at least 95% or 90% identical thereto:
i) SEQ ID NO.: 501 and SEQ ID NO.:502;
ii) SEQ ID NO.: 504 and SEQ ID NO.:505;
iii) SEQ ID NO.: 507 and SEQ ID NO.:508;
iv) SEQ ID NO.: 510 and SEQ ID NO.:511;
v) SEQ ID NO.: 513 and SEQ ID NO.:514;
vi) SEQ ID NO.: 516 and SEQ ID NO.:517; and
vii) SEQ ID NO.: 519 and SEQ ID NO.:520; or
l. introducing a nucleic acid sequence encoding a TCR α chain and a TCR β chain polypeptide of (k).

### Claim at position 20

23-29. (canceled)

### Claim 30 (depends on 22)

The method of claim 22, wherein the introduced nucleic acid sequence, or the first nucleic acid sequence and the second nucleic acid sequence, are introduced via a vector, via transfection, via a lipid nanoparticle, or via microinjection.

### Claim 31

(canceled)

### Claim 32

A method of in locus insertion of a TCR, optionally a WT1-specific TCR, comprising delivering to a cell (i) a first guide RNA for inserting the TCR that comprises a guide sequence selected from: SEQ ID NOs: 90, 95, 97, 98, 185, 214, and 218, (ii) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent; and (iii) a donor nucleic acid molecule that encodes a TCR, optionally a WT1-specific TCR.

### Claim 33 (depends on 32)

The method of claim 32, further comprising delivering a second guide RNA comprising a sequence selected from SEQ ID NOs: 1-89.

### Claim at position 25

34-35. (canceled)

### Claim 36

A composition comprising:
a. a guide RNA comprising
i. a guide sequence selected from SEQ ID NOs: 1-89; or
ii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-89; or
iii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 1-89; or
iv. a guide sequence comprising any one of SEQ ID NOs: 1-24; or
v. a guide sequence comprising any one of SEQ ID NOs: 1-6; or
vi. a guide sequence selected from SEQ ID NOs: 90-178, 185, and 213-218; or
vii. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218; or
viii. a guide sequence that is at least 95% or 90% identical to a sequence selected from SEQ ID NOs: 90-178, 185, and 213-218; or
ix. a guide sequence comprising any one of SEQ ID NOs: 90-113 and 213-218; or
v. a guide sequence comprising any one of SEQ ID NOs: 90-95; and
b. an RNA-guided DNA binding agent or nucleic acid encoding an RNA-guided DNA binding agent.

### Claim at position 27

37-43. (canceled)

### Claim 44 (depends on 5)

A cell, altered by the method of claim 5.

### Claim at position 29

45-48. (canceled)

### Claim 49 (depends on 44)

The cell according to claim 44, lacking an endogenous T-cell receptor, for preparation of a T cell expressing a non-endogenous T-cell receptor.

### Claim at position 31

50-53. (canceled)

### Claim 54 (depends on 44)

A cell according to claim 44, additionally comprising one or more nucleic acid sequences encoding α and β chains of an exogenous T-cell receptor.

### Claim 55 (depends on 54)

A cell of claim 54, wherein the one or more nucleic acid sequences encoding the α and β chains of the exogenous T-cell receptor are in the TRAC locus of the genome.

### Claim at position 34

56-58. (canceled)

### Claim 59 (depends on 54)

A cell of claim 54, wherein the TCR α chain is encoded by a nucleic acid sequence according to any of SEQ ID NOs: 500, 501, 503, and 504, and the β TCR chain is encoded according to any of SEQ ID NOs: 500, 502, 503, and 505.

### Claim at position 36

60-63. (canceled)

### Claim 64 (depends on 44)

A population of cells comprising cells of claim 44, wherein greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 98%, or greater than about 99% of the altered population are CD3− cells.

### Claim at position 38

65-99. (canceled)

### Claim 100 (depends on 1)

The method of claim 1,
a) wherein the guide RNA comprises a guide sequence selected from any one of SEQ ID NOs 1-178, 185, and 213-218 and further comprises
i) a nucleotide sequence of SEQ ID NO: 400, wherein the nucleotides of SEQ ID NO: 400 follow the guide sequence at its 3′ end; or
ii) a nucleotide sequence of SEQ ID NO: 401, wherein the nucleotides of SEQ ID NO: 401 follow the guide sequence at its 3′ end; or
b) wherein the guide RNA is modified according to the pattern of SEQ ID NO: 300, wherein the N's are collectively any one of the guide sequences of SEQ ID NOs 1-89, 185, and 213-218.

### Claim at position 40

101-122. (canceled)

### Claim 123 (depends on 1)

The method of claim 1, wherein the RNA-guided DNA binding agent is Cas9.

### Claim 124

(canceled)

### Claim 125 (depends on 1)

The method of claim 1, wherein the composition is a pharmaceutical formulation and further comprises a pharmaceutically acceptable carrier.

### Claim at position 44

126-322. (canceled)

### Claim 323 (depends on 36, 44)

A method of treating a human or animal subject comprising administering the composition of claim 36 or the cell of claim 44, wherein the method treats a disease or disorder, wherein the disease or disorder is cancer.

### Claim 324 (depends on 323)

The method of claim 323, wherein the cancer is characterized by tumors that overexpress Wilms' tumor antigen (WT1).