T-cell modulatory multimeric polypeptides and methods of use thereof
The present disclosure provides variant immunomodulatory polypeptides, and fusion polypeptides comprising the variant immunomodulatory peptides. The present disclosure provides T-cell modulatory multimeric polypeptides, and compositions comprising same, where the T-cell modulatory multimeric polypeptides comprise a variant immunomodulatory polypeptide of the present disclosure. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the T-cell modulatory multimeric polypeptides, and host cells comprising the nucleic acids. The present disclosure provides methods of modulating the activity of a T cell; the methods comprise contacting the T cell with a T-cell modulatory multimeric polypeptide of the present disclosure.
1. A nucleic acid comprising:
a) a first nucleotide sequence encoding a first polypeptide comprising:
i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO:13); and
ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2-microglobulin (β2M) polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:17, and
b) a second nucleotide sequence encoding a second polypeptide comprising:
i) two copies of an IL-2 polypeptide, each copy comprising an amino acid sequence set forth in SEQ ID NO:84;
ii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:19; and
iii) an immunoglobulin (Ig) Fc polypeptide.
2. A nucleic acid of claim 1 ,
wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% percent amino acid sequence identity to SEQ ID NO:57, wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST, and wherein the Ig Fc polypeptide comprises an L14A substitution and an L15A substitution based on the amino acid numbering depicted in SEQ ID NO:57,
wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide, and
wherein the second polypeptide comprises a peptide linker between one or more of:
a) a first copy of the IL-2 polypeptide and a second copy of the IL-2 polypeptide;
b) one of the two copies of the IL-2 polypeptide and the MHC class I heavy chain polypeptide; and
c) the MHC class I heavy chain polypeptide and the Ig Fc polypeptide.
3. An expression vector comprising the nucleic acid of claim 1 .
4. An expression vector comprising the nucleic acid of claim 2 .
5. A plurality of cells genetically modified with the expression vector of claim 3 .
6. A plurality of cells genetically modified with the expression vector of claim 4 .
7. A plurality of cells genetically modified with the nucleic acid of claim 1 .
8. A plurality of cells genetically modified with the nucleic acid of claim 2 .
9. A method comprising culturing a plurality of genetically modified cells according to claim 5 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
10. A method comprising culturing a plurality of genetically modified cells according to claim 6 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
11. A method comprising culturing a plurality of genetically modified cells according to claim 7 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
12. A method comprising culturing a plurality of genetically modified cells according to claim 8 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
13. First and second nucleic acids, wherein
a) the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide comprising:
i) a human papillomavirus epitope, wherein the epitope comprises the amino acid sequence YMLDLQPETT (SEQ ID NO:13); and
ii) a first major histocompatibility complex (MHC) polypeptide, wherein the first MHC polypeptide is a β2-microglobulin (β2M) polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:17, and
b) the second nucleic acid comprises a second nucleotide sequence encoding a second polypeptide comprising:
i) two copies of an IL-2 polypeptide, each copy comprising an amino acid sequence set forth in SEQ ID NO:84;
ii) a second MHC polypeptide, wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:19; and
iii) an immunoglobulin (Ig) Fc polypeptide.
14. The nucleic acids of claim 13 ,
wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% percent amino acid sequence identity to SEQ ID NO:57, wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST, and wherein the Ig Fc polypeptide comprises an L14A substitution and an L15A substitution based on the amino acid numbering depicted in SEQ ID NO:57,
wherein the first polypeptide comprises a peptide linker between the epitope and the β2M polypeptide, and
wherein the second polypeptide comprises a peptide linker between one or more of:
a) a first copy of the IL-2 polypeptide and a second copy of the IL-2 polypeptide;
b) one of the two copies of the IL-2 polypeptide and the MHC class I heavy chain polypeptide; and
c) the MHC class I heavy chain polypeptide and the Ig Fc polypeptide.
15. One or more expression vectors comprising the nucleic acids of claim 13 .
16. One or more expression vectors comprising the nucleic acids of claim 14 .
17. A plurality of cells genetically modified with the one or more expression vectors of claim 15 .
18. A plurality of cells genetically modified with the one or more expression vectors of claim 16 .
19. A plurality of cells genetically modified with the nucleic acids of claim 13 .
20. A plurality of cells genetically modified with the nucleic acids of claim 14 .
21. A method comprising culturing a plurality of genetically modified cells according to claim 17 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
22. A method comprising culturing a plurality of genetically modified cells according to claim 18 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
23. A method comprising culturing a plurality of genetically modified cells according to claim 19 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.
24. A method comprising culturing a plurality of genetically modified cells according to claim 20 in a culture medium under conditions such that the cell synthesizes the first and second polypeptides.