MODIFIED ALLOGENEIC CELLS AND METHODS AND COMPOSITIONS FOR THE PREPARATION THEREOF
The present disclosure features allogeneic modified cells (e.g., T- or NK-cells) having increased persistence, increased resistance to immune rejection, or decreased risk of eliciting a host-versus-graft reaction, or a combination thereof. Methods for producing and using the same are also provided.
1 . A fusion polypeptide comprising an HLA heavy chain polypeptide, or a functional fragment thereof, and a beta-2-microglobulin (B2M) polypeptide, or a functional fragment thereof.
2 . The fusion polypeptide of claim 1 , wherein the HLA heavy chain polypeptide and/or the B2M polypeptide comprise an amino acid alteration that reduces affinity of binding to a CD8 polypeptide relative to a wild-type HLA heavy chain polypeptide and/or B2M polypeptide.
3 . The fusion polypeptide of claim 1 , wherein the HLA heavy chain polypeptide is an HLA-A, HLA-B, or HLA-C polypeptide.
4 . The fusion polypeptide of claim 3 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations to one or more of amino acid positions 183-274.
5 . The fusion polypeptide of claim 4 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations at an amino acid position selected from the group consisting of A73, D227, T228, and A245.
6 . The fusion polypeptide of any one of claims 1-5 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations selected from the group consisting of A73T, D227K, T228A, and A245V.
7 . The fusion polypeptide of claim 6 , wherein the HLA heavy chain polypeptide comprises the alterations D227K and T228A, or the alterations D227K, T228A, and A245V.
8 . The fusion polypeptide of any one of claims 1-7 , wherein the B2M polypeptide comprises an amino acid alteration.
9 . The fusion polypeptide of claim 8 , wherein the B2M polypeptide comprises an amino acid alteration at position K58.
10 . The fusion polypeptide of claim 9 , wherein the B2M polypeptide comprises the amino acid alteration K58E.
11 . The fusion polypeptide of claim 1 further comprising a cognate peptide (cPep).
12 . The fusion polypeptide of claim 11 , wherein the cPep comprises from about 8 to about 10 amino acids.
13 . The fusion polypeptide of claim 12 , wherein the cPep comprises 9 amino acids.
14 . The fusion polypeptide of claim 12 or 13 , wherein the cPep comprises an amino acid sequence selected from the group consisting of QYDDAVYKL (SEQ ID NO: 520), RYRPGTVAL (SEQ ID NO: 521), LSSPVTKSF (SEQ ID NO: 522), EEVHDLERKY (SEQ ID NO: 523), RLRAEAQVK (SEQ ID NO: 524), IIDKSGAAV (SEQ ID NO: 529; IV9 (AA)), IIDKSGEEV (SEQ ID NO: 530; IV9 (EE)), IIDKSGLAV (SEQ ID NO: 531; IV9 (LA)), IIDKSGSTV (SEQ ID NO: 532; IV9 (WT)), and the cPep sequences listed in Table A.
15 . The fusion polypeptide of any one of claims 1-14 , wherein the fusion polypeptide comprises one or more linkers.
16 . The fusion polypeptide of claim 15 , wherein the one or more linkers comprise the amino acid sequence (GGGGS) n (SEQ ID NO: 247).
17 . The fusion polypeptide of claim 16 , wherein n is 3 or 4.
18 . The fusion polypeptide any one of claims 1-17 further comprising a signal peptide.
19 . The fusion polypeptide of claim 18 , wherein the signal peptide is a B2M signal peptide.
20 . The fusion polypeptide of claim 19 , wherein the signal peptide comprises the amino acid sequence MSRSVALAVLALLSLSGLEA (SEQ ID NO: 525).
21 . The fusion polypeptide of any one of claims 1-20 , further comprising a transmembrane domain.
22 . The fusion polypeptide of claim 21 , wherein the transmembrane domain is a cluster of differentiation 4 (CD4) transmembrane (CD4TM) domain.
23 . The fusion polypeptide of claim 22 , wherein the CD4TM domain comprises an amino acid sequence with at least about 85% sequence identity to the amino acid sequence MALIVLGGVAGLLLFIGLGIFFCVRC (SEQ ID NO: 437).
24 . The fusion polypeptide of any one of claims 1-23 , wherein the HLA heavy chain polypeptide comprises a sequence with at least 85% sequence identity to an amino acid sequence selected from the group consisting of:
HLA-A*02 (HLA-A2)
(SEQ ID NO: 441)
GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGET
RKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKE
DLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHH
AVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQR
YTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSY
SQAASSDSAQGSDVSLTACKV;
HLA-A*03
(SEQ ID NO: 442)
GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQET
RNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNE
DLRSWTAADMAAQITKRKWEAAHEAEQLRAYLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHH
PISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSY
TQAASSDSAQGSDVSLTACKV;
HLA-B*57
(SEQ ID NO: 445)
GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDGET
RNMKASAQTYRENLRIALRYYNQSEAGSHIIQVMYGCDVGPDGRLLRGHDQSAYDGKDYIALNE
DLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHH
PISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSY
SQAACSDSAQGSDVSLTA;
HLA-B*44
(SEQ ID NO: 446)
GSHSMRYFYTAMSRPGRGEPRFITVGYVDDTLFVRFDSDATSPRKEPRAPWIEQEGPEYWDRET
QISKTNTQTYRENLRTALRYYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQDAYDGKDYIALNE
DLSSWTAADTAAQITQRKWEAARVAEQDRAYLEGLCVESLRRYLENGKETLQRADPPKTHVTHH
PISDHEVTLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSY
SQAACSDSAQGSDVSLTA;
HLA-C*04
(SEQ ID NO: 449)
GSHSMRYFSTSVSWPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPREPWVEQEGPEYWDRET
QKYKRQAQADRVNLRKLRGYYNQSEDGSHTLQRMFGCDLGPDGRLLRGYNQFAYDGKDYIALNE
DLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHH
PVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPEPLTLRWKPSSQPTIPIVGIVAGLAVLAVLAVLGAMVAVVMCRRKSSGGKGGS
CSQAASSNSAQGSDESLIACKA;
HLA-C*05
(SEQ ID NO: 450)
CSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRET
QKYKRQAQTDRVNLRKLRGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNE
DLRSWTAADKAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHH
PVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPEPLTLRWGPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGS
CSQAASSNSAQGSDESLIACKA;
and
HLA-C*07
(SEQ ID NO: 451)
CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRET
QKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNE
DLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHH
PLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQR
YTCHMQHEGLQEPLTLSWEPSSQPTIPIMGIVAGLAVLVVLAVLGAVVTAMMCRRKSSGGKGGS
CSQAACSNSAQGSDESLITCKA.
25 . The fusion polypeptide of any one of claims 1-24 , wherein the B2M polypeptide comprises a sequence with at least 85% sequence identity to the amino acid sequence
(SEQ ID NO: 426)
IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV
EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRD
M.
26 . The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises from N-terminus to C-terminus:
A) the B2M polypeptide and the HLA heavy chain polypeptide;
B) the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
C) a cognate peptide (cPep), the B2M polypeptide, and the HLA heavy chain polypeptide;
D) a cognate peptide (cPep), the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
E) a cognate peptide (cPep), a linker, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
F) a signal peptide, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
G) a signal peptide, the B2M polypeptide and the HLA heavy chain polypeptide;
H) a signal peptide, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
I) a signal peptide, a cognate peptide (cPep), the B2M polypeptide, and the HLA heavy chain polypeptide;
J) a signal peptide, a cognate peptide (cPep), the B2M polypeptide, a linker, and the HLA heavy chain polypeptide; or
K) a signal peptide, a cognate peptide (cPep), a linker, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide.
27 . The fusion polypeptide of claim 26 further comprising a transmembrane domain C-terminal to the HLA heavy chain polypeptide.
28 . A fusion polypeptide comprising an amino acid sequence with at least 85% sequence identity to a polypeptide sequence listed in Table 8A.
29 . A polynucleotide encoding the fusion polypeptide of any one of claims 1-26 .
30 . The polynucleotide of claim 29 , wherein the polynucleotide comprises a sequence with at least 85% sequence identity to a polynucleotide sequence listed in Table 8B.
31 . A vector comprising a polynucleotide encoding the fusion polypeptide of any one of claims 1-26 .
32 . The vector of claim 31 , wherein the vector is a viral vector or a transposon.
33 . The vector of claim 32 , wherein the vector is a lentiviral vector.
34 . A cell comprising the fusion polypeptide of any one of claims 1-28 , the polynucleotide of claim 29 or claim 30 , or the vector of any one of claims 31-33 .
35 . The cell of claim 34 , wherein the cell is an immune cell, a hepatocyte, a stem cell, an induced pluripotent stem cell, an islet cell, or a progenitor thereof.
36 . A pharmaceutical composition comprising the polynucleotide of claim 29 or claim 30 , the vector of any one of claims 31-33 , or the cell of claim 34 or claim 35 , and a pharmaceutically acceptable excipient.
37 . A method for preparing a modified cell, the method comprising:
a) modifying a cell to knock-out expression of an endogenous beta-2-microglobulin (B2M) polypeptide in the cell; and
b) contacting the cell with a polynucleotide encoding a fusion polypeptide comprising an HLA heavy chain polypeptide, or a functional fragment thereof, and a beta-2-microglobulin (B2M) polypeptide, or a functional fragment thereof, and expressing the polypeptide in the cell.
38 . The method of claim 37 , wherein the HLA heavy chain polypeptide and/or the B2M polypeptide comprise an amino acid alteration that that reduces affinity of binding to a CD8 polypeptide relative to a wild-type HLA heavy chain polypeptide and/or B2M polypeptide.
39 . The method of claim 37 , wherein the HLA heavy chain polypeptide is an HLA-A, HLA-B, HLA-C, or HLA-E polypeptide.
40 . The method of claim 37 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations to one or more of amino acid positions 183-274.
41 . The method of claim 37 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations at an amino acid position selected from the group consisting of A73, D227, T228, and A245.
42 . The method of any one of claims 37-41 , wherein the HLA heavy chain polypeptide comprises one or more amino acid alterations selected from the group consisting of A73T, D227K, T228A, and A245V.
43 . The method of claim 42 , wherein the HLA heavy chain polypeptide comprises the alterations D227K and T228A, or the alterations D227K, T228A, and A245V.
44 . The method of any one of claims 37-43 , wherein the B2M polypeptide comprises an amino acid alteration.
45 . The method of claim 44 , wherein the B2M polypeptide comprises an amino acid alteration at position K58.
46 . The method of claim 45 , wherein the B2M polypeptide comprises the amino acid alteration K58E.
47 . The method of claim 37 wherein the fusion polypeptide further comprises a cognate peptide (cPep).
48 . The method of claim 47 , wherein the cPep comprises from about 8 to about 10 amino acids.
49 . The method of claim 48 , wherein the cPep comprises 9 amino acids.
50 . The method of claim 48 or claim 49 , wherein the cPep comprises an amino acid sequence selected from the group consisting of QYDDAVYKL (SEQ ID NO: 520), RYRPGTVAL (SEQ ID NO: 521), LSSPVTKSF (SEQ ID NO: 522), EEVHDLERKY (SEQ ID NO: 523), RLRAEAQVK (SEQ ID NO: 524), IIDKSGAAV (SEQ ID NO: 529; IV9 (AA)), IIDKSGEEV (SEQ ID NO: 530; IV9 (EE)), IIDKSGLAV (SEQ ID NO: 531; IV9 (LA)), IIDKSGSTV (SEQ ID NO: 532; IV9 (WT)), and the cPep sequences listed in Table A.
51 . The method of any one of claims 37-50 , wherein fusion polypeptide comprises one or more linkers.
52 . The method of claim 51 , wherein the one or more linkers comprise the amino acid sequence (GGGGS) n (SEQ ID NO: 247).
53 . The method of claim 52 , wherein n is 3 or 4.
54 . The method any one of claims 37-53 , wherein the fusion polypeptide further comprises a signal peptide.
55 . The method of claim 54 , wherein the signal peptide is a B2M signal peptide.
56 . The method of claim 55 , wherein the signal peptide comprises the amino acid sequence MSRSVALAVLALLSLSGLEA (SEQ ID NO: 525).
57 . The method of any one of claims 37-56 , wherein the fusion polypeptide further comprises a transmembrane domain.
58 . The method of claim 57 , wherein the transmembrane domain is a cluster of differentiation 4 (CD4) transmembrane (CD4TM) domain.
59 . The method of claim 58 , wherein the CD4TM domain comprises an amino acid sequence with at least about 85% sequence identity to the amino acid sequence
(SEQ ID NO: 437)
MALIVLGGVAGLLLFIGLGIFFCVRC.
60 . The method of any one of claims 37-59 , wherein the HLA heavy chain polypeptide comprises a sequence with at least 85% sequence identity to an amino acid sequence selected from the group consisting of:
HLA-A*02 (HLA-A2)
(SEQ ID NO: 441)
GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGET
RKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKE
DLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHH
AVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQR
YTCHVQHEGLPKPLTLRWEPSSQPTIPIVGIIAGLVLFGAVITGAVVAAVMWRRKSSDRKGGSY
SQAASSDSAQGSDVSLTACKV;
HLA-A*03
(SEQ ID NO: 442)
GSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDQET
RNVKAQSQTDRVDLGTLRGYYNQSEAGSHTIQIMYGCDVGSDGRFLRGYRQDAYDGKDYIALNE
DLRSWTAADMAAQITKRKWEAAHEAEQLRAYLDGTCVEWLRRYLENGKETLQRTDPPKTHMTHH
PISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWELSSQPTIPIVGIIAGLVLLGAVITGAVVAAVMWRRKSSDRKGGSY
TQAASSDSAQGSDVSLTACKV;
HLA-B*57
(SEQ ID NO: 445)
GSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRMAPRAPWIEQEGPEYWDGET
RNMKASAQTYRENLRIALRYYNQSEAGSHIIQVMYGCDVGPDGRLLRGHDQSAYDGKDYIALNE
DLSSWTAADTAAQITQRKWEAARVAEQLRAYLEGLCVEWLRRYLENGKETLQRADPPKTHVTHH
PISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSY
SQAACSDSAQGSDVSLTA;
HLA-B*44
(SEQ ID NO: 446)
GSHSMRYFYTAMSRPGRGEPRFITVGYVDDTLFVRFDSDATSPRKEPRAPWIEQEGPEYWDRET
QISKTNTQTYRENLRTALRYYNQSEAGSHIIQRMYGCDVGPDGRLLRGYDQDAYDGKDYIALNE
DLSSWTAADTAAQITQRKWEAARVAEQDRAYLEGLCVESLRRYLENGKETLQRADPPKTHVTHH
PISDHEVTLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQR
YTCHVQHEGLPKPLTLRWEPSSQSTVPIVGIVAGLAVLAVVVIGAVVAAVMCRRKSSGGKGGSY
SQAACSDSAQGSDVSLTA;
HLA-C*04
(SEQ ID NO: 449)
GSHSMRYFSTSVSWPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPREPWVEQEGPEYWDRET
QKYKRQAQADRVNLRKLRGYYNQSEDGSHTLQRMFGCDLGPDGRLLRGYNQFAYDGKDYIALNE
DLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKETLQRAEHPKTHVTHH
PVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPEPLTLRWKPSSQPTIPIVGIVAGLAVLAVLAVLGAMVAVVMCRRKSSGGKGGS
CSQAASSNSAQGSDESLIACKA;
HLA-C*05
(SEQ ID NO: 450)
CSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVQFDSDAASPRGEPRAPWVEQEGPEYWDRET
QKYKRQAQTDRVNLRKLRGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYNQFAYDGKDYIALNE
DLRSWTAADKAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRYLENGKKTLQRAEHPKTHVTHH
PVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQR
YTCHVQHEGLPEPLTLRWGPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGS
CSQAASSNSAQGSDESLIACKA;
and
HLA-C*07
(SEQ ID NO: 451)
CSHSMRYFDTAVSRPGRGEPRFISVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRET
QKYKRQAQADRVSLRNLRGYYNQSEDGSHTLQRMSGCDLGPDGRLLRGYDQSAYDGKDYIALNE
DLRSWTAADTAAQITQRKLEAARAAEQLRAYLEGTCVEWLRRYLENGKETLQRAEPPKTHVTHH
PLSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQR
YTCHMQHEGLQEPLTLSWEPSSQPTIPIMGIVAGLAVLVVLAVLGAVVTAMMCRRKSSGGKGGS
CSQAACSNSAQGSDESLITCKA.
61 . The method of any one of claims 37-60 , wherein the B2M polypeptide comprises a sequence with at least 85% sequence identity to the amino acid sequence
(SEQ ID NO: 426)
IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKV
EHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRD
M.
62 . The method of claim 37 , wherein the fusion polypeptide comprises from N-terminus to C-terminus:
A) the B2M polypeptide and the HLA heavy chain polypeptide;
B) the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
C) a cognate peptide (cPep), the B2M polypeptide, and the HLA heavy chain polypeptide;
D) a cognate peptide (cPep), the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
E) a cognate peptide (cPep), a linker, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
F) a signal peptide, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
G) a signal peptide, the B2M polypeptide and the HLA heavy chain polypeptide;
H) a signal peptide, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide;
I) a signal peptide, a cognate peptide (cPep), the B2M polypeptide, and the HLA heavy chain polypeptide;
J) a signal peptide, a cognate peptide (cPep), the B2M polypeptide, a linker, and the HLA heavy chain polypeptide; or
K) a signal peptide, a cognate peptide (cPep), a linker, the B2M polypeptide, a linker, and the HLA heavy chain polypeptide.
63 . The method of claim 52 further comprising a transmembrane domain C-terminal to the HLA heavy chain polypeptide.
64 . The method of any one of claims 37-63 , further comprising contacting the cell with a vector comprising the polynucleotide.
65 . The method of claim 64 , wherein the vector is a viral vector or a transposon.
66 . The method of claim 65 , wherein the vector is a lentiviral vector.
67 . The method of any one of claims 37-66 , wherein modifying the cell to knock-out expression of B2M is carried out using base editing.
68 . The method of claim 67 , wherein the base editing comprises contacting the cell with a base editor and a guide polynucleotide targeting the base editor to effect an alteration of a nucleobase of an endogenous B2M gene in the cell, thereby knocking out expression of the endogenous B2M polypeptide in the cell.
69 . The method of claim 68 , wherein the base editor is a cytidine deaminase base editor, an adenosine deaminase base editor, or a cytidine adenosine deaminase base editor.
70 . The method of claim 69 , wherein the adenosine deaminase is TadA or a TadA variant.
71 . The method of claim 70 , wherein the TadA variant is a TadA*8 or TadA*9.
72 . The method of claim 69 , wherein the cytidine deaminase is APOBEC or an APOBEC variant.
73 . The method of claim 69 , wherein the deaminase is TadA*8.20.
74 . The method of any one of claims 68-73 , wherein the guide polynucleotide comprises a spacer comprising the nucleotide sequence CUUACCCCACUUAACUAUCU (SEQ ID NO: 537).
75 . The method of any one of claims 68-74 , wherein the guide polynucleotide comprises a scaffold comprising the nucleotide sequence
(SEQ ID NO: 626)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA
ACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.
76 . The method of any one of claims 37-75 , further comprising expressing a chimeric antigen receptor in the cell.
77 . The method of claim 76 , wherein the chimeric antigen receptor targets an antigen expressed on the surface of a neoplastic cell.
78 . The method of any one of claims 37-60 , wherein a natural killer cell contacted with the modified cell shows reduced levels of granulation relative to levels measured when the natural killer cell is contacted with a reference cell.
79 . The method of any one of claims 37-78 , wherein the cell is a T cell, a natural killer cell, a hepatocyte, a stem cell, an induced pluripotent stem cell, an islet cell, or a progenitor thereof.
80 . The method of claim 79 , wherein the cell is a CD4+ or CD8+ cell.
81 . The method of any one of claims 37-80 , wherein the immune cell is an allogeneic cell.
82 . The method of any one of claims 37-81 , wherein the modified cell shows increased resistance to lysis by an immune effector cell relative to a reference cell.
83 . The method of claim 82 , wherein lysis is reduced by at least about 60%.
84 . The method of claim 82 , wherein lysis is reduced by at least about 90%.
85 . The method of any one of claims 37-84 , wherein an alloreactive T cell contacted with the modified cell shows reduced levels of cytokine production relative to levels produced when the alloreactive T cell is contacted with a reference cell.
86 . The method of claim 85 , wherein granzyme B (GZMB) secreted by the alloreactive T cell is reduced by at least about 10%.
87 . The method of claim 85 or claim 86 , wherein granzyme B (GZMB) secreted by the alloreactive T cell is reduced by at least about 50%.
88 . The method of any one of any one of claims 85-87 , wherein INF-gamma secreted by the alloreactive T cell is reduced by at least about 10%.
89 . The method of any one of claims 85-88 , wherein INF-gamma secreted by the alloreactive T cell is reduced by at least about 40%.
90 . The method any one of claims 85-88 , wherein TNF-alpha secreted by the alloreactive T cell is reduced by at least about 10%.
91 . The method any one of claims 85-88 , wherein TNF-alpha secreted by the alloreactive T cell is reduced by at least about 40%.
92 . The method of any one of claims 37-91 , wherein the modified cell further comprises virtually undetectable levels of HLA-A and HLA-B.
93 . A method for preparing a modified cell, the method comprising:
a) modifying a cell to knock-out expression of an endogenous beta-2-microglobulin (B2M) polypeptide in the cell; and
b) contacting the cell with a polynucleotide encoding a B2M polypeptide, and expressing the B2M polypeptide in the cell, wherein
i) expression of the B2M polypeptide is reduced relative to an unmodified cell; and/or
ii) the B2M polypeptide comprises an alteration at one or more amino acids positions that effects a reduction in binding of the B2M polypeptide to an HLA heavy chain polypeptide
94 . The method of claim 93 , wherein the B2M polypeptide is expressed in the cell under the control of a promoter.
95 . The method of claim 94 , wherein the promoter is an EF1a promoter.
96 . The method of any one of claims 93-95 , wherein the B2M polypeptide comprises an alteration at amino acid position K58, W60, and/or position W95.
97 . The method of claim 96 , wherein the B2M polypeptide comprises a W60G and/or a W95G amino acid alteration.
98 . The method of any one of claims 93-97 , wherein modifying the cell to knock-out expression of B2M is carried out using base editing.
99 . The method of claim 98 , wherein the base editing comprises contacting the cell with a base editor and a guide polynucleotide targeting the base editor to effect an alteration of a nucleobase of an endogenous B2M gene in the cell, thereby knocking out expression of the endogenous B2M polypeptide in the cell.
100 . The method of claim 99 , wherein the base editor is a cytidine deaminase base editor, an adenosine deaminase base editor, or a cytidine adenosine deaminase base editor.
101 . The method of claim 100 , wherein the adenosine deaminase is TadA or a TadA variant.
102 . The method of claim 101 , wherein the TadA variant is a TadA*8 or TadA*9.
103 . The method of claim 100 , wherein the cytidine deaminase is APOBEC or an APOBEC variant.
104 . The method of claim 100 , wherein the deaminase is TadA*8.20.
105 . The method of any one of claims 99-104 , wherein the guide polynucleotide comprises a spacer comprising the nucleotide sequence CUUACCCCACUUAACUAUCU (SEQ ID NO: 537).
106 . The method of any one of claims 99-105 , wherein the guide polynucleotide comprises a scaffold comprising the nucleotide sequence
(SEQ ID NO: 626)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.
107 . The method of any one of claims 93-106 , further comprising expressing a chimeric antigen receptor in the cell.
108 . The method of claim 107 , wherein the chimeric antigen receptor targets an antigen expressed on the surface of a neoplastic cell.
109 . The method of any one of claims 93-108 , wherein a natural killer cell contacted with the modified cell shows reduced levels of granulation relative to levels measured when the natural killer cell is contacted with a reference cell.
110 . The method of any one of claims 93-109 , wherein the cell is a T cell, a natural killer cell, a hepatocyte, a stem cell, an induced pluripotent stem cell, an islet cell, or a progenitor thereof.
111 . The method of claim 110 , wherein the cell is a CD4+ or CD8+ cell.
112 . The method of any one of claims 93-111 , wherein the cell is an allogeneic cell.
113 . The method of any one of claims 93-112 , wherein the modified immune cell shows increased resistance to lysis by an immune effector cell relative to a reference cell.
114 . The method of claim 113 , wherein lysis is reduced by at least about 60%.
115 . The method of claim 113 or claim 114 , wherein lysis is reduced by at least about 90%.
116 . The method of any one of claims 93-115 , wherein an alloreactive T cell contacted with the modified cell shows reduced levels of cytokine production relative to levels produced when the alloreactive T cell is contacted with a reference cell.
117 . The method of claim 116 , wherein granzyme B (GZMB) secreted by the alloreactive T cell is reduced by at least about 10%.
118 . The method of claim 116 or claim 117 , wherein granzyme B (GZMB) secreted by the alloreactive T cell is reduced by at least about 50%.
119 . The method of any one of claims 116-118 , wherein INF-gamma secreted by the alloreactive T cell is reduced by at least about 10%.
120 . The method of any one of claims 116-119 , wherein INF-gamma secreted by the alloreactive T cell is reduced by at least about 40%.
121 . The method any one of claims 116-120 , wherein TNF-alpha secreted by the alloreactive T cell is reduced by at least about 10%.
122 . The method any one of claims 116-121 , wherein TNF-alpha secreted by the alloreactive T cell is reduced by at least about 40%.
123 . The method of any one of claims 93-122 , wherein the method effects a reduction in levels of surface expression of an HLA-A, HLA-B, and/or HLA-C polypeptide on the cell relative to a reference cell.
124 . The method of claim 123 , wherein surface expression is reduced by at least about 40%.
125 . The method of claim 123 , wherein surface expression is reduced by at least about 80%.
126 . The method of any one of claims 93-125 , wherein the modified cell further comprises virtually undetectable levels of HLA-A and HLA-B.
127 . The method of any one of claims 93-126 , wherein expression of the B2M polypeptide is reduced by at least about 75% relative to an unmodified cell.
128 . The method of any one of claims 93-127 , wherein expression of the B2M polypeptide is reduced by at least about 90% relative to an unmodified cell.
129 . A modified cell prepared according to the method of any one of claims 37-128 .
130 . The modified cell of claim 129 , wherein the cell expresses a chimeric antigen receptor that binds an antigen expressed on the surface of a neoplastic cell.
131 . The modified cell of claim 130 , wherein a natural killer cell contacted with the modified cell shows reduced levels of granulation relative to levels measured when the natural killer cell is contacted with a reference cell.
132 . A pharmaceutical composition comprising the modified cell of any one of claims 129-131 and a pharmaceutically acceptable excipient.
133 . A method for killing a neoplastic cell, the method comprising contacting the neoplastic cell with the cell of any one of claim 33-35 or 129-131 , or the pharmaceutical composition of claim 36 or claim 132 .
134 . A method for treating a subject having a neoplasia, the method comprising administering to the subject the cell of any one of claim 33-35 or 129-131 , or the pharmaceutical composition of claim 36 or claim 132 .
135 . A kit for use in the method of any one of claim 37-128, 133, or 134 , wherein the kit comprises the fusion polypeptide, polynucleotide, vector, pharmaceutical composition, and/or cell of any of the above claims .