Genetically Modified Cells Expressing Antigen-Containing Fusion Proteins and Uses Thereof
The application relates to biological components, methods, systems, and kits for modulating immune responses. The disclosed biological components include genetically modified cells comprising an inserted exogenous sequence in a major histocompatibility complex (MHC)-associated gene. The inserted exogenous sequence encodes a peptide and the genetically modified cells express a fusion protein comprising the peptide and at least a portion of the polypeptide encoded by the MHC-associated gene to form a modified MHC complex. The genetically modified cells may present the peptide as an antigen associated with the MHC complex and may be utilized in methods for modulating T cell activity, inducing an immune response, and inducing a tolerogenic response. As such, the disclosed biological components, methods, systems, and kits may be utilized in order to treat and/or prevent a disease or disorder in a subject in need thereof and to screen and validate clinically relevant antigens.
1 . A cell comprising a genetically modified major histocompatibility complex (MHC) associated gene, wherein the genetically modified MHC-associated gene has been genetically modified to comprise an inserted polynucleotide sequence encoding a peptide and optionally a linker such that the genetically modified MHC-associated gene encodes a fusion protein comprising the peptide and optional linker.
2 . The cell of claim 1 , wherein the peptide comprises a non-self antigen, a self-antigen, or a neoantigen.
3 . (canceled)
4 . (canceled)
5 . The cell of claim 1 , wherein the linker comprises 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acids select from G, S, and A, and optionally comprises (G 4 S) n where n is selected from 3-6.
6 . (canceled)
7 . The cell of claim 1 , wherein the cell is capable of presenting the peptide via the MHC to a T cell to induce activation or tolerance and/or the cell presents the peptide via the MHC to a T cell to induce activation or tolerance.
8 . The cell of claim 1 , wherein the inserted polynucleotide sequence encodes a fusion protein comprising the peptide and optionally a linker fused in-frame to at least a portion of a coding sequence of an MHC-associated gene and the inserted polynucleotide sequence knocks-out an endogenous MHC-associated gene.
9 . The cell of claim 8 , wherein the endogenous MHC-associated gene is a B2M gene.
10 . The cell of claim 1 , wherein the inserted polynucleotide sequence does not comprise an MHC-associated gene and the peptide and the optional linker are inserted in-frame with a coding sequence of an endogenous MHC-associated gene.
11 . The cell of claim 1 , wherein the peptide does not comprise a signal peptide encoded by an MHC-associated gene.
12 . The cell of claim 1 , wherein the B2M gene in the cell is not knocked out.
13 . The cell of claim 1 , wherein the inserted polynucleotide sequence produces a homologous knock-in in the cell.
14 . The cell claim 1 , wherein the MHC-associated gene is the B2M gene.
15 . The cell of claim 14 , wherein the inserted polynucleotide sequence is inserted in a region of the B2M gene that encodes for the N-terminus of the B2M protein or the C-terminus of the B2M protein.
16 . The cell of claim 14 , wherein the inserted polynucleotide sequence is inserted in a region of the B2M gene that encodes for the first twenty amino acids encoding region of the B2M protein.
17 . The cell of claim 1 , wherein the genetically modified MHC-associated gene has been further genetically modified to comprise a second inserted polynucleotide sequence encoding a second MHC-associated gene and optionally a linker such that the genetically modified MHC-associated gene encodes a fusion protein comprising the peptide and optionally the linker.
18 . The cell of claim 17 , wherein the second inserted polynucleotide sequence is inserted in a region that encodes for the B2M protein.
19 . The cell of claim 17 , wherein the second inserted polynucleotide sequence is inserted in a region of the B2M gene that encodes for the C-terminus of the B2M protein.
20 . The cell of claim 1 , wherein the MHC-associated gene is an MHC class I gene.
21 . The cell of claim 20 , wherein the MHC class I gene is an HLA class I gene.
22 . The cell of claim 21 , wherein the HLA class I gene is a gene encoding an HLA-A protein, an HLA-B protein, an HLA-C protein, an HLA-E protein, an HLA-L protein, an HLA-J protein, an HLA-K protein, an HLA-H protein, or an HLA-G protein.
23 . (canceled)
24 . The cell of claim 1 , wherein the MHC-associated gene is a B2M gene encoding a B2M protein having a N-terminus and a C-terminus, wherein the inserted polynucleotide sequence encoding the peptide is inserted in frame with the N-terminus of the B2M protein and optionally further comprising a second inserted polynucleotide encoding a second peptide linked in frame to the C-terminus of the B2M protein and wherein the second polynucleotide encoding the second peptide is further optionally linked in frame to a polynucleotide encoding a MHC class I protein.
25 . The cell of claim 1 , wherein the cell is an antigen presenting cell (APC) or an artificial antigen presenting cell (aAPC).
26 . The cell of claim 25 , wherein the APC is a dendritic cell, a macrophage, a monocyte or a B cell.
27 . (canceled)
28 . (canceled)
29 . The cell of claim 1 , wherein the cell is an autologous or allogeneic cell relative to a recipient subject to which the cell may be administered.
30 . (canceled)
31 . A method of activating a T cell comprising contacting the T cell with the cell of claim 1 .
32 . A method of inducing tolerance in a T cell, the method comprising contacting the T cell with the cell of claim 1 .
33 . (canceled)
34 . A method of the cell of claim 1 , the method comprising introducing into the cell:
a. a CRISPR effector protein or a polynucleotide encoding a CRISPR effector protein;
b. a guide polynucleotide comprising a guide sequence designed to hybridize with a target sequence in the MHC-associated gene in the cell; and
c. a donor polynucleotide comprising a polynucleotide sequence encoding the peptide;
wherein the CRISPR effector protein introduces a double-stranded break at the target sequence and repair of the double-stranded break through a DNA repair process results in insertion of the inserted polynucleotide sequence encoding the peptide in the MHC-associated gene in the cell thereby producing a modified cell expressing a genetically modified MHC-associated gene, wherein the genetically modified MHC-associated gene encodes a fusion protein comprising the peptide.
35 . (canceled)
36 . A method of modulating an immune response in a subject comprising administering the cell of claim 1 to the subject.
37 . A method of modulating an immune response in a subject comprising administering a population of activated or tolerogenic T cells to the subject, wherein the population of activated or tolerogenic T cells are produced by contacting the cell of claim 1 with a T cell or a population of T cells.
38 .- 47 . (canceled)
48 . The method of claim 34 , wherein the CRISPR effector protein is a type II or type V CRISPR effector protein.
49 .- 51 . (canceled)
52 . The method of claim 34 , wherein the CRISPR effector protein is Cas14a, a Cas14b, or a Cas14c polypeptide.
53 .- 62 . (canceled)
63 . The method of claim 31 , wherein the T cell is an effector T cell, a cytotoxic T cell, a helper T cell, or a regulatory T cell.
64 .- 87 . (canceled)
88 . The method of claim 36 , wherein the subject is a human.
89 . (canceled)
90 . A system comprising:
a. a CRISPR effector protein or a polynucleotide encoding a CRISPR effector protein;
b. a guide polynucleotide comprising a guide sequence designed to hybridize with a target sequence in an MHC-associated gene in a cell; and
c. a donor polynucleotide comprising a polynucleotide sequence encoding a peptide for genetically modifying the MHC-associated gene via insertion of the donor polynucleotide at the MHC-associated gene in frame with the protein encoded by the MHC-associated gene, wherein the genetically modified MHC-associated gene encodes a fusion protein comprising the peptide.
91 .- 121 . (canceled)