PERSISTENT ALLOGENEIC MODIFIED IMMUNE CELLS AND METHODS OF USE THEREOF

1 . A method for producing a persistent allogeneic modified immune cell, the method comprising contacting a cell with a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp), a deaminase, and one or more guide RNAs (gRNAs) that target the base editor to effect an alteration in a nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of HLA-A, HLA-B, HLA-C, Transporter Associated with Antigen Processing I (TAP1), Transporter Associated with Antigen Processing II (TAP2), Tapasin/TAP Binding Protein (TAPBP), TAP-Binding Protein-Like (TAPBPL), NLR family CARD domain containing 5 (NLRC5)/MHC class I transactivator (CITA), cluster of differentiation 155 (CD155), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB) polypeptide, nectin cell adhesion molecule 2 (Nectin-2), and UL16 binding protein 1-6 (ULBP), thereby producing the persistent allogeneic modified immune cell.

2 . The method of claim 1 , wherein the method further comprises contacting the cell with one or more guide RNAs that target the base editor to effect an alteration in a nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of beta-2 microglobulin, CD48, CD58, Protein Disulfide Isomerase Family A Member 3 (PDIA3/ERp57), and T Cell Receptor Alpha Constant polypeptides.

3 . The method of claim 1 , wherein the method comprises effecting a nucleobase alteration that reduces expression on the cell of a polypeptide selected from the group consisting of HLA-A, HLA-B, and HLA-C.

4 . The method of claim 1 , wherein the one or more gRNAs comprise a nucleotide sequence with at least about 85% sequence identity to GCACUCACCCGCCCAGGUCU (SEQ ID NO: 817; TSBTx4190), GACCCGCAUCUCGGCGUCUG (SEQ ID NO: 827; TSBTx4200), CCUUACCCCAUCUCAGGGUG (SEQ ID NO: 820; TSBTx4193), and/or CUUACCCCAUCUCAGGGUGA (SEQ ID NO: 821; TSBTx4194).

5 . The method of claim 1 , further comprising overexpressing in the cell an inhibitory receptor, or fragment thereof, selected from the group consisting of Human Leukocyte Antigen-E (HLA-E), Human Leukocyte Antigen-G (HLA-G), Programmed Death Ligand 1 (PD-L1), and Cluster of Differentiation 47 (CD47).

6 . A method for producing a persistent allogeneic modified immune cell, the method comprising:

(a) contacting a cell with a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp), a deaminase, and one or more guide RNAs (gRNAs) that target a nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide or comprises a regulatory element associated with expression of the polypeptide, and wherein the polypeptide is selected from the group consisting of HLA-A, HLA-B, HLA-C, Transporter Associated with Antigen Processing I (TAP1), Transporter Associated with Antigen Processing II (TAP2), Tapasin/TAP Binding Protein (TAPBP), TAP-Binding Protein-Like (TAPBPL), NLR family CARD domain containing 5 (NLRC5)/MHC class I transactivator (CITA), cluster of differentiation 155 (CD155), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB) polypeptide, nectin cell adhesion molecule 2 (Nectin-2), and UL16 binding protein 1-6 (ULBP); and

(b) overexpressing in the cell an inhibitory receptor, or fragment thereof, selected from the group consisting of Human Leukocyte Antigen-E (HLA-E), Human Leukocyte Antigen-G (HLA-G), Programmed Death Ligand 1 (PD-L1), and Cluster of Differentiation 47 (CD47).

7 . An allogeneic modified immune cell produced according to the method of claim 1 .

8 . An allogeneic modified immune cell comprising a nucleobase alteration that reduces or eliminates expression of a polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, Transporter Associated with Antigen Processing I (TAP1), Transporter Associated with Antigen Processing II (TAP2), Tapasin/TAP Binding Protein (TAPBP), TAP-Binding Protein-Like (TAPBPL), NLR family CARD domain containing 5 (NLRC5)/MHC class I transactivator (CITA), cluster of differentiation 155 (CD155), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB) polypeptide, nectin cell adhesion molecule 2 (Nectin-2), and UL16 binding protein 1-6 (ULBP).

9 . A pharmaceutical composition comprising an effective amount an allogeneic modified immune cell of claim 8 .

10 . A composition comprising a guide RNA (gRNA) and a polynucleotide encoding a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp) domain and a deaminase domain, wherein the gRNA comprises a nucleic acid sequence that is complementary to a polynucleotide, wherein the polynucleotide encodes a polypeptide or comprises a regulatory element associated with expression of the polypeptide, wherein the polypeptide is selected from the group consisting of HLA-A, HLA-B, HLA-C, Transporter Associated with Antigen Processing I (TAP1), Transporter Associated with Antigen Processing II (TAP2), Tapasin/TAP Binding Protein (TAPBP), TAP-Binding Protein-Like (TAPBPL), NLR family CARD domain containing 5 (NLRC5)/MHC class I transactivator (CITA), cluster of differentiation 155 (CD155), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB) polypeptide, nectin cell adhesion molecule 2 (Nectin-2), and UL16 binding protein 1-6 (ULBP).

11 . The composition of claim 10 , wherein the guide RNA comprises a nucleotide sequence selected from the group consisting of GCACUCACCCGCCCAGGUCU (SEQ ID NO: 817; TSBTx4190), GACCCGCAUCUCGGCGUCUG (SEQ ID NO: 827; TSBTx4200), CCUUACCCCAUCUCAGGGUG (SEQ ID NO: 820; TSBTx4193), and CUUACCCCAUCUCAGGGUGA (SEQ ID NO: 821; TSBTx4194).

12 . A kit comprising an allogeneic modified immune cell of claim 8 .

13 . A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of an allogeneic modified immune cell of claim 8 .

14 . A fusion polypeptide comprising a loading peptide, at least a fragment of an HLA-G polypeptide, and at least a fragment of a β2M polypeptide.

15 . The fusion polypeptide of claim 14 , wherein the recombinant polypeptide comprises from N-terminus to C-terminus:

a) a loading peptide, at least a fragment of an HLA-G polypeptide, and at least a fragment of a β2M polypeptide;

b) at least a fragment of a β2M polypeptide, a loading peptide, and at least a fragment of an HLA-G polypeptide;

c) a loading peptide, at least a fragment of a β2M polypeptide, and at least a fragment of an HLA-G polypeptide; or

d) fragment of an HLA-G polypeptide, a loading peptide, and at least at least a fragment of a β2M polypeptide.

16 . A fusion polypeptide comprising a loading peptide, at least a fragment of an HLA-E polypeptide, and at least a fragment of a β2M polypeptide.

17 . A fusion polypeptide comprising a loading peptide, and at least a fragment of an HLA-E polypeptide.

18 . A fusion polypeptide comprising an amino acid sequence with at least 85% sequence identity to a sequence selected from the group consisting of:

HLA-G5 + IL-2 signal peptide

(SEQ ID NO: 1013)

MYRMQLLSCIALSLALVTNSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDD

TQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQT

LRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDL

RSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQ

RADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVEL

VETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDG

GIMSVRESRSLSEDL; 

HLA-G5 Single chain trimer + IL-2 signal peptide

(SEQ ID NO: 1014)

MYRMQLLSCIALSLALVTNSIQRTPKIQVYSRHPAENGKSNFLNCYVSGF

HPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYAC

RVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSRIIPRHLQLGGGGSGG

GGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFD

SDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQ

SEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAAD

TAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKT

HVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAG

DGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRE

SRSLSEDL; 

HLA-E(ΔTM) Single chain trimer + HLA-G5 intron 

tail

(SEQ ID NO: 1015)

MSRSVALAVLALLSLSGLEAVMAPRTLFLGGGGSGGGGSGGGGSIQRTPK

IQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF

SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGG

GGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFD

NDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQ

SEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVD

TAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKT

HVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAG

DGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWSKEGDGGIMSVRE

SRSLSEDL; 

HLA-E(ΔTM) β2M (C-term) Single chain trimer

(SEQ ID NO: 1016)

MSRSVALAVLALLSLSGLEAVMAPRTLFLGGSGGGASGGGSHSLKYFHTS

VSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWD

RETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLR

GYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLE

DTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPA

EITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHV

QHEGLPEPVTLRWGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNF

LNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTP

TEKDEYACRVNHVTLSQPKIVKWDRDM; 

HLA-E(ΔTM) Single chain dimer + HLA-G5 intron 

tail

(SEQ ID NO: 1017)

MSRSVALAVLALLSLSGLEAVMAPRTLFLGGSGGGASGGGSHSLKYFHTS

VSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWD

RETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLR

GYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLE

DTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPA

EITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHV

QHEGLPEPVTLRWSKEGDGGIMSVRESRSLSEDL; 

and

HLA-E(ΔTM) Single chain dimer

(SEQ ID NO: 1018)

MSRSVALAVLALLSLSGLEAVMAPRTLFLGGSGGGASGGGSHSLKYFHTS

VSRPGRGEPRFISVGYVDDTQFVRFDNDAASPRMVPRAPWMEQEGSEYWD

RETRSARDTAQIFRVNLRTLRGYYNQSEAGSHTLQWMHGCELGPDGRFLR

GYEQFAYDGKDYLTLNEDLRSWTAVDTAAQISEQKSNDASEAEHQRAYLE

DTCVEWLHKYLEKGKETLLHLEPPKTHVTHHPISDHEATLRCWALGFYPA

EITLTWQQDGEGHTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHV

QHEGLPEPVTLRW.

19 . A membrane-bound fusion polypeptide, wherein the fusion polypeptide comprises a β2M domain, and an HLA-E domain and/or a transmembrane domain.

20 . A fusion polypeptide comprising an amino acid sequence having at least 85% sequence identity to the following sequence:

(SEQ ID NO: 1019)

MSRSVALAVLALLSLSGLEAVMAPRTLFLGGGGSGGGGSGGGGSIQRTPK

IQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSF

SKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGG

GGSGGGGSGGGGSGSHSLKYFHTSVSRPGRGEPRFISVGYVDDTQFVRFD

NDAASPRMVPRAPWMEQEGSEYWDRETRSARDTAQIFRVNLRTLRGYYNQ

SEAGSHTLQWMHGCELGPDGRFLRGYEQFAYDGKDYLTLNEDLRSWTAVD

TAAQISEQKSNDASEAEHQRAYLEDTCVEWLHKYLEKGKETLLHLEPPKT

HVTHHPISDHEATLRCWALGFYPAEITLTWQQDGEGHTQDTELVETRPAG

DGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPVTLRWKPASQPTIPIMAL

IVLGGVAGLLLFIGLGIFFCVRC.

21 . A mammalian expression vector comprising a polynucleotide sequence encoding the fusion polypeptide of claim 14 .

22 . An allogeneic modified immune cell comprising the vector of claim 21 .

23 . A method for producing a persistent allogeneic modified immune cell, the method comprising contacting a cell with a polynucleotide programmable DNA binding polypeptide (napDNAbp) and one or more guide RNAs (gRNAs) that target the napDNAbp to cleave a target nucleic acid molecule and introduce an alteration in the target nucleic acid molecule, wherein the target nucleic acid molecule encodes a polypeptide and/or comprises a regulatory element associated with expression thereof, and wherein the polypeptide is selected from the group consisting of HLA-A, HLA-B, HLA-C, Transporter Associated with Antigen Processing I (TAP1), Transporter Associated with Antigen Processing II (TAP2), Tapasin/TAP Binding Protein (TAPBP), TAP-Binding Protein-Like (TAPBPL), NLR family CARD domain containing 5 (NLRC5)/MHC class I transactivator (CITA), cluster of differentiation 155 (CD155), MHC class I polypeptide-related sequence A (MICA), MHC class I polypeptide-related sequence B (MICB) polypeptide, nectin cell adhesion molecule 2 (Nectin-2), and UL16 binding protein 1-6 (ULBP), thereby producing the persistent allogeneic modified immune cell.

24 . A method for producing a persistent allogeneic modified immune cell, the method comprising contacting a cell with a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp), a deaminase, and guide RNAs (gRNAs) that target the base editor to effect an alteration in one or more nucleic acid molecules, wherein the one or more nucleic acid molecules encode the following polypeptides and/or comprise regulatory elements associated with expression thereof: CD5, B2M, CD3 gamma, CD3 epsilon, CIITA, and PD-1 (PD1), thereby producing the persistent allogeneic modified immune cell; or contacting a cell with a base editor comprising a polynucleotide programmable DNA binding polypeptide (napDNAbp), a deaminase, and guide RNAs (gRNAs) that target the base editor to effect an alteration in one or more nucleic acid molecules, wherein the one or more nucleic acid molecules encode the following polypeptides and/or comprise regulatory elements associated with expression thereof: HLA-A, HLA-B, and CIITA, thereby producing the persistent allogeneic modified immune cell, wherein the persistent allogeneic modified immune cell surface-expresses HLA-C.

25 . An allogeneic modified immune cell produced by the method of claim 24 .

26 . A method of treating cancer in a subject, the method comprising administering to the subject an effective amount of the allogeneic modified immune cell of claim 25 .