Peptide markers to track genetically engineered cells
To allow control over injected genetically engineered cells, it is helpful that the genetically engineered cells express a marker that can be used to detect such cells among a pool of unmodified cells. Some embodiments relate to a marked protein comprising a TCR constant domain and an exogenous amino acid variation that comprises a sequence that is detectable and identifiable within the TCR constant domain. Other embodiments relate to an antibody epitope that is attached to a TCR chain. Both the marked proteins and the antibody epitopes can be used to track genetically engineered cells.
1 . A marked protein comprising:
a human TCR constant domain, wherein the TCR constant domain comprises a TCRα or TCRβ constant domain; and
an exogenous amino acid variation that comprises a sequence that is detectable and identifiable within the TCR constant domain, wherein the exogenous amino acid variation is from a non-human species,
wherein the exogenous amino acid variation consists of 1 to 10 amino acid mutations, and wherein at least one of the mutations is in a FG loop.
2 . The marked protein of claim 1 , wherein the non-human species is a mouse.
3 . The marked protein of claim 1 , wherein the TCR constant domain comprises a sequence encoded by a human TRBC2 gene.
4 . The marked protein of claim 1 , wherein the exogenous amino acid variation comprises a sequence of a murine TCR Cβ domain.
5 . The marked protein of claim 4 , wherein the exogenous amino acid variation consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid mutations selected from the group consisting of: K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P, as numbered according to the numbering system of SEQ ID NO: 8.
6 . The marked protein of claim 4 , wherein the exogenous amino acid variation comprises 6 amino acid mutations.
7 . The marked protein of claim 6 , wherein the 6 amino acid mutations are: K4R, E108K, T110P, Q111E, D112G, and R113S, as numbered according to the numbering system of SEQ ID NO: 8.
8 . The marked protein of claim 1 , wherein the exogenous amino acid variation is detectable and identifiable by an antibody, a single-domain antibody, a Fab fragment or a designed ankyrin repeat protein.
9 . The marked protein of claim 1 , wherein the exogenous amino acid variation is detectable and identifiable by an anti-mouse TCR Cβ antibody H57-597.
10 . A genetically engineered cell comprising the marked protein of claim 1 .
11 . The marked protein of claim 1 , wherein the marked protein comprises huTRBC2-mur6 (SEQ ID NO: 27), huTRBC2-mur7 (SEQ ID NO: 26), huTRBC2-mur10 (SEQ ID NO: 15), or any one of SEQ ID NOs: 17-19 or 25.
12 . The marked protein of claim 1 , wherein the marked protein comprises huTRBC1-mur6 or huTRBC1-muFG (SEQ ID NO: 14).
13 . The marked protein of claim 1 , wherein the exogenous amino acid variation consists of 6, 7, 8, 9, or 10 amino acid mutations,
wherein the marked protein comprises human TCRβC1 and the mutations are selected from the group consisting of: N4R, F7T, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P, as numbered according to the numbering system of SEQ ID NO: 11, or
wherein the marked protein comprises human TCRβC2 and the mutations are selected from the group consisting of: K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P, as numbered according to the numbering system of SEQ ID NO: 8.
14 . The marked protein of claim 13 , wherein the marked protein comprises human TCRβC1 and the exogenous amino acid variation consists of N4R, E108K, T110P, Q111E, D112G, and R113S, as numbered according to the numbering system of SEQ ID NO: 11.
15 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, E108K, T110P, Q111E, D112G, and R113S, as numbered according to the numbering system of SEQ ID NO: 8.
16 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, E108K, T110P, Q111E, D112G, and R113S, and 1, 2, 3 or 4 additional mutations selected from the group consisting of: F7T, Y37F, N106E and A114P, as numbered according to the numbering system of SEQ ID NO: 8.
17 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, Y37F, E108K, T110P, Q111E, D112G, and R113S as numbered according to the numbering system of SEQ ID NO: 8.
18 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, and R113S, as numbered according to the numbering system of SEQ ID NO: 8.
19 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P as numbered according to the numbering system of SEQ ID NO: 8.
20 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, F7T, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P as numbered according to the numbering system of SEQ ID NO: 8.
21 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, F7T, Y37F, E108K, T110P, Q111E, D112G, R113S, and A114P as numbered according to the numbering system of SEQ ID NO: 8.
22 . The marked protein of claim 13 , wherein the exogenous amino acid variation consists of K4R, F7T, Y37F, N106E, E108K, T110P, Q111E, D112G, R113S, and A114P as numbered according to the numbering system of SEQ ID NO: 8.