MODIFIED IMMUNE CELLS HAVING ADENOSINE DEAMINASE BASE EDITORS FOR MODIFYING A NUCLEOBASE IN A TARGET SEQUENCE
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.
1 . A base editor system for producing a modified immune cell, base editor system comprising a nucleobase editor polypeptide, or a polynucleotide encoding the nucleobase editor polypeptide, and 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.
2 . The system of claim 1 , wherein the adenosine deaminase variant domain comprises both an alteration at amino acid position 82 and the T166R amino acid alteration.
3 . The system of claim 1 , wherein the adenosine deaminase variant domain comprises a V82S alteration and/or the T166R amino acid alteration.
4 . The system of claim 1 , wherein the adenosine deaminase variant domain comprises one or more of the following alterations: 176Y, Y147T, Y147R, Q154S, Y123H, and Q154R.
5 . The system 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+176Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+176Y; V82S+Y123H+Y147R+Q154R; and I76Y+V82S+Y123H+Y147R+Q154R.
6 . The system 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.
7 . The system 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
8 . The system 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.
9 . The system of claim 1 , wherein the napDNAbp comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 22.
10 . The system 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.
11 . The system of claim 1 , wherein the nucleobase editor polypeptide further comprises a linker between the napDNAbp and the adenosine deaminase variant domain.
12 . The system of claim 1 , wherein the nucleobase editor polypeptide further comprises one or more nuclear localization signals (NLS).
13 . The system of claim 1 , wherein the modified immune cell is a T cell.
14 . The system of claim 1 , further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).
15 . A modified immune cell produced by contacting an immune cell with the system of claim 1 .
16 . The modified immune cell of claim 15 , wherein the modified immune 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).
17 . The modified immune cell of claim 15 , wherein the immune cell expresses a chimeric antigen receptor.
18 . The modified immune cell of claim 17 , wherein the chimeric antigen receptor comprises an extracellular domain having an affinity for a marker associated with neoplasia.
19 . The modified immune cell of claim 18 , wherein the neoplasia is a B cell cancer, a lymphoma, a leukemia, or multiple myeloma.
20 . 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 the base editor system of claim 14 .