MULTIPLEXED IMMUNOHISTOCHEMISTRY USING RECOMBINANT ANTIBODIES WITH EPITOPE TAGS
The present disclosure is directed to epitope-tagged antibodies, as well as methods of employing the epitope-tagged antibodies for detecting one or more targets in a biological sample, e.g. a tissue sample.
1 . An epitope-tagged antibody comprising an antibody and one or more epitope tag constructs, wherein each epitope tag construct of the one or more epitope tag constructs comprises one or more peptide-based epitope tags separated by one or more peptide-based spacers, wherein each epitope tag construct has the formula:
-[Spacer] a -[Epitope Tag] b -,
where a and b are each independently an integer ranging from 1 to 10.
2 . The epitope-tagged antibody of claim 1 , wherein the one or more epitope tag constructs are expressed at a C-terminal end of a heavy chain constant region of the antibody and/or at a C-terminal end of a light chain constant region of the antibody.
3 . The epitope-tagged antibody of claim 2 , wherein the one or more epitope tag constructs are expressed at both the C-terminal end of the heavy chain constant region of the antibody and at the C-terminal end of the light chain constant region of the antibody.
4 . The epitope-tagged antibody of claim 3 , wherein a ratio of a number of epitope tags incorporated at the C-terminal end of a heavy chain constant region to a number of epitope tags incorporated at the C-terminal end of a light chain constant region ranges from 2:1 to about 1:2.
5 . The epitope-tagged antibody of claim 3 , wherein a number of epitope tags incorporated at the C-terminal end of the heavy chain constant region ranges from between 2 to 6 epitope tags, and a number of epitope tags incorporated at the C-terminal end of the light chain constant region ranges from between 0 to 4 epitope tags.
6 . The epitope-tagged antibody of claim 1 , wherein the epitope-tagged antibody comprises two epitope tag constructs, wherein each epitope tag construct is expressed at a terminal end of a heavy chain of the antibody, and wherein each epitope tag construct comprises four epitope tags.
7 . The epitope-tagged antibody of claim 1 , wherein a combined molecular weight of all epitope tag constructs of any epitope-tagged antibody is less than about 30% of the molecular weight of the corresponding native antibody.
8 . The epitope-tagged antibody of claim 1 , wherein a distance between successive epitope tags in each epitope tag construct of the one or more epitope constructs is less than about 18 nm.
9 . The epitope-tagged antibody of claim 1 , wherein a distance between successive epitope tags in each epitope tag construct of the one or more epitope constructs is between about 8 mm and about 18 mm.
10 . The epitope-tagged antibody of claim 1 , wherein at least a portion of an amino acid sequence constituting at least one spacer of the one or more epitope tag constructs is selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14.
11 . The epitope-tagged antibody of claim 1 , wherein each epitope tag of the one or more epitope tag constructs comprises a sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
12 . The epitope-tagged antibody of claim 1 , wherein each epitope tag construct of the one or more epitope constructs epitope tag construct comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOS: 16, 18, 20, 22, 24, 26, 28, 30, and 32.
13 . The epitope-tagged antibody of claim 1 , wherein each of the one or more peptide-based spacers is encoded by a nucleotide sequence having between 20 and 60 nucleotides.
14 . The epitope-tagged antibody of claim 1 , wherein each epitope tag construct of the one or more epitope constructs comprises between 2 and 8 epitope tags.
15 . The epitope-tagged antibody of claim 1 , wherein the epitope tag is selected from the group consisting of V5, HA, VSV, AU1, AU5, OLLAS, E, E2, and KT3.
16 . An epitope tag construct comprising an amino acid sequence having at least 90% identity to any one of SEQ ID NOS: 16, 18, 20, 22, 24, 26, 28, 30, and 32.
17 . An antibody expressing at least one epitope tag construct, the epitope tag construct comprising alternating spacers and epitope tags; wherein the epitope tag comprises an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9; wherein at least one of the spacers comprises an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14; and wherein the antibody expresses between about 2 to about 8 epitope tags.
18 . The antibody of claim 17 , wherein the antibody expresses between about 2 to about 6 epitope tags.
19 . The antibody of claim 17 , wherein the antibody comprises two epitope tag constructs.
20 . The antibody of claim 17 , wherein the antibody has a molecular weight ranging from about 170 kDa to about 270 kDa.