# US 12,600,971 B2 · Modified immune cells having adenosine deaminase base editors for modifying a nucleobase in a target sequence

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- Number: US 12,600,971 B2
- Document ID: US12600971B2
- Status: Granted
- Kind: B2
- Application number: 17/430,676
- Filed: 2020-02-13
- Published: 2023-03-16
- Granted: 2026-04-14
- Earliest claimed: 2019-02-13
- Assignees: Beam Therapeutics Inc.
- Applicants: Beam Therapeutics Inc.
- Inventors: Nicole Gaudelli; Michael Packer; Ian Slaymaker; Yi Yu; Bernd Zetsche; David A. Born; Seung-Joo Lee; Jason M. Gehrke
- Web page: https://patentgrep.com/patent/US12600971B2

## CPC

- C12N 15/1138: {against receptors or cell surface proteins}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/315: Phosphorothioates
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2320/33: Alteration of splicing
- C12N 2510/00: Genetically modified cells
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present invention features genetically modified immune cells comprising novel adenosine base editors (e.g., ABE8) having enhanced anti-neoplasia activity, resistance to immune suppression, and decreased risk of eliciting a graft-versus-host reaction or host-versus-graft reaction, or a combination thereof. The present invention also features methods for producing and using these modified immune effector cells.

## Claims

### Claim 1

A method for producing a modified immune cell, the method comprising expressing or introducing in an immune cell a nucleobase editor polypeptide and contacting the cell with two or more guide RNAs that target the nucleobase editor polypeptide to effect an alteration in a nucleic acid molecule encoding at least one polypeptide selected from the group consisting of a T Cell Receptor Alpha Constant (TRAC), beta-2 microglobulin (B2M), programmed cell death 1 (PD1), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 5 (CD5), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 123 (CD123), Cbl Proto-Oncogene B (CBLB), and Class II Major Histocompatibility Complex Transactivator (CIITA) polypeptide, wherein the nucleobase editor polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and at least one base editor domain comprising an adenosine deaminase variant domain comprising an alteration at amino acid position 82 of SEQ ID NO. 3 and/or comprising an amino acid alteration referenced to SEQ ID NO: 3 selected from the group consisting of I76Y, Y147T, Y147R, Q154S, and T166R.

### Claim 2 (depends on 1)

The method of claim 1, wherein the adenosine deaminase variant domain comprises both an alteration at amino acid position 82 and the T166R amino acid alteration.

### Claim 3 (depends on 1)

The method of claim 1, wherein the adenosine deaminase variant domain comprises a V82S alteration and/or the T166R amino acid alteration.

### Claim 4 (depends on 1)

The method of claim 1, wherein the adenosine deaminase variant domain further comprises one or more of the following alterations: I76Y, Y147T, Y147R, Q154S, Y123H, and Q154R.

### Claim 5 (depends on 1)

The method of claim 1, wherein the adenosine deaminase variant domain comprises a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.

### Claim 6 (depends on 1)

The method of claim 1, wherein the adenosine deaminase variant is selected from the group consisting of TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, and TadA*8.24.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleobase editor polypeptide is selected from the group consisting of ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, and ABE8.24-m.

### Claim 8 (depends on 1)

The method of claim 1, wherein the base editor domain is an adenosine deaminase variant heterodimer comprising a wild-type adenosine deaminase domain and the adenosine deaminase variant domain.

### Claim 9 (depends on 1)

The method of claim 1, wherein the napDNAbp comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 22.

### Claim 10 (depends on 1)

The method of claim 1, wherein the napDNAbp is a Staphylococcus aureus Cas9 (SaCas9), a Streptococcus thermophilus 1 Cas9 (StlCas9), a Streptococcus pyogenes Cas9 (SpCas9), or variants thereof.

### Claim 11 (depends on 1)

The method of claim 1, wherein the nucleobase editor polypeptide further comprises a linker between the napDNAbp and the adenosine deaminase variant domain.

### Claim 12 (depends on 1)

The method of claim 1, wherein the nucleobase editor polypeptide further comprises one or more nuclear localization signals (NLS).

### Claim 13 (depends on 1)

The method of claim 1, wherein the immune cell is a T cell.

### Claim 14 (depends on 1)

The method of claim 1, wherein the nucleobase editor polypeptide further comprises one or more uracil glycosylase inhibitors.

### Claim 15 (depends on 1)

The method of claim 1, further comprising expressing a chimeric antigen receptor (CAR) in the modified immune cell.

### Claim 16 (depends on 1)

A modified immune cell produced according to the method of claim 1.

### Claim 17 (depends on 16)

The modified immune cell of claim 16, wherein the cell comprises one or more mutations in polynucleotides encoding B2M, CD7, CIITA, PD1, CBLB, and/or TRAC;
TIGIT, TGFBR2, ZAP70, NFATc1, or TET2; or
V-Set Immunoregulatory Receptor (VISTA), T Cell Immunoglobulin Mucin 3 (Tim-3), T Cell Immunoreceptor With Ig and ITIM Domains (TIGIT), Transforming Growth Factor Beta Receptor II (TGFbRII), Regulatory Factor X Associated Ankyrin Containing Protein (RFXANK), PVR Related Immunoglobulin Domain Containing (PVRIG), Lymphocyte-Activation Gene 3 (Lag3), Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), Chitinase 3 Like 1 (Chi311), Cluster of Differentiation 96 (CD96), B and T Lymphocyte Associated (BTLA), Tet Methylcytosine Dioxygenase 2 (TET2), Sprouty RTK Signaling Antagonist 1 (Spry1), Sprouty RTK Signaling Antagonist 2 (Spry2), Class II Major Histocompatibility Complex Transactivator (CIITA), Cluster of Differentiation 7 (CD7), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 52 (CD52), Cluster of Differentiation 123 (CD123), T Cell Receptor Beta Constant 1 (TRBC1), T Cell Receptor Beta Constant 2 (TRBC2), Cytokine Inducible SH2 Containing Protein (CISH), Acetyl-CoA Acetyltransferase 1 (ACAT1), Cytochrome P450 Family 11 Subfamily A Member 1 (Cyp11a1), GATA Binding Protein 3 (GATA3), Nuclear Receptor Subfamily 4 Group A Member 1 (NR4A1), Nuclear Receptor Subfamily 4 Group A Member 2 (NR4A2), Nuclear Receptor Subfamily 4 Group A Member 3 (NR4A3), Methylation-Controlled J Protein (MCJ), Fas Cell Surface Death Receptor (FAS), or Selectin P Ligand/P-Selectin Glycoprotein Ligand-1 (SELPG/PSGL1).

### Claim 18 (depends on 16)

The modified immune cell of claim 16, wherein the immune cell expresses a chimeric antigen receptor.

### Claim 19 (depends on 18)

The modified immune cell of claim 18, wherein the chimeric antigen receptor comprises an extracellular domain having an affinity for a marker associated with neoplasia.

### Claim 20 (depends on 19)

The modified immune cell of claim 19, wherein the neoplasia is a B cell cancer, a lymphoma, a leukemia, or multiple myeloma.

### Claim 21 (depends on 16)

A method of modulating an immune response, treating a neoplasia or in a subject or treating graft-versus-host disease (GVHD) in a subject having or having a propensity to develop graft-versus-host disease, the method comprising administering an effective amount of a modified immune cell of claim 16.

### Claim 22 (depends on 16)

A pharmaceutical composition or kit comprising an effective amount a modified immune cell of claim 16 in a pharmaceutically acceptable excipient.