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 cancer-associated epitope other than a human papillomavirus epitope; and
ii) a β2-microglobulin (β2M) polypeptide;
b) a second nucleotide sequence encoding a second polypeptide comprising:
i) a major histocompatibility complex (MHC) class I HLA-A heavy chain polypeptide;
ii) two copies of a variant IL-2 polypeptide, each copy comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 is other than a His and amino acid 42 is other than a Phe, and wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST; and
iii) an immunoglobulin (Ig) Fc polypeptide.
2. A nucleic acid of claim 1 ,
wherein the β2M polypeptide comprises an amino acid sequence having at least 95% percent amino acid sequence identity to amino acids 21 to 119 of SEQ ID NO:95,
wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% percent amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:57,
wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST, and
wherein amino acid 16 in each of the variant IL-2 polypeptides is Ala, Glu, Thr, or Asp,
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 components of the second polypeptide.
3. The nucleic acid of claim 2 , wherein the Ig Fc polypeptide comprises an L14A substitution and an L15A substitution based on the amino acid numbering set forth in SEQ ID NO:57, and
wherein (i) the β2M polypeptide comprises a Cys residue at amino acid 12 of the β2M polypeptide and (ii) the MHC class I heavy chain polypeptide comprises a Cys at residue 236, based on the numbering set forth in SEQ ID NO:19.
4. The nucleic acid of claim 2 , wherein each variant IL-2 polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 in each of the variant IL-2 polypeptides is Ala, Glu, Thr, or Asp, and amino acid 42 is Ala, wherein the two variant IL-2 polypeptides are linked by a peptide linker, and wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST.
5. The nucleic acid of claim 2 , wherein the MHC class I heavy chain polypeptide in each heterodimer is an HLA-A, HLA-B, HLA-C or HLA-E heavy chain polypeptide.
6. The nucleic acid of claim 5 , 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 (i) the β2M polypeptide comprises a Cys residue at amino acid 12 of the β2M polypeptide and (ii) the MHC class I heavy chain polypeptide comprises a Cys at residue 236, based on the numbering set forth in SEQ ID NO:19, and
wherein each variant IL-2 polypeptide has the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 is Ala and amino acid 42 is Ala.
7. A plurality of cells genetically modified with the nucleic acid of claim 2 .
8. A plurality of cells genetically modified with the nucleic acid of claim 6 .
9. 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.
10. 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.
11. First and second nucleic acids, wherein
a) the first nucleic acid comprises a nucleotide sequence encoding a first polypeptide comprising:
i) a cancer-associated epitope other than a human papillomavirus epitope; and
ii) a β2-microglobulin (β2M) polypeptide;
b) a second nucleotide sequence encoding a second polypeptide comprising:
i) a major histocompatibility complex (MHC) class I HLA-A heavy chain polypeptide;
ii) two copies of a variant IL-2 polypeptide, each copy comprising an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 is other than a His and amino acid 42 is other than a Phe, and wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST; and
iii) an immunoglobulin (Ig) Fc polypeptide.
12. The nucleic acids of claim 11 , wherein the β2M polypeptide comprises an amino acid sequence having at least 95% percent amino acid sequence identity to amino acids 21 to 119 of the amino acid sequence set forth in SEQ ID NO:95,
wherein the Ig Fc polypeptide comprises an amino acid sequence having at least about 95% percent amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:57,
wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST, and
wherein amino acid 16 in each of the variant IL-2 polypeptides is Ala, Glu, Thr, or Asp,
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 components of the second polypeptide.
13. The nucleic acids of claim 12 , wherein the Ig Fc polypeptide comprises an L14A substitution and an L15A substitution based on the amino acid numbering set forth in SEQ ID NO:57, and
wherein (i) the β2M polypeptide comprises a Cys residue at amino acid 12 of the β2M polypeptide and (ii) the MHC class I heavy chain polypeptide comprises a Cys at residue 236, based on the numbering set forth in SEQ ID NO:19.
14. The nucleic acids of claim 12 , wherein each variant IL-2 polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 in each of the variant IL-2 polypeptides is Ala, Glu, Thr, or Asp, and amino acid 42 is Ala, wherein the two variant IL-2 polypeptides are linked by a peptide linker, and wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST.
15. The nucleic acids of claim 12 , wherein the MHC Class I heavy chain polypeptide in each heterodimer is an HLA-A, HLA-B, HLA-C or HLA-E heavy chain polypeptide.
16. The nucleic acids of claim 15 ,
wherein the Ig Fc polypeptide comprises an L14A substitution and an L15A substitution based on the amino acid numbering set forth in SEQ ID NO:57,
wherein (i) the β2M polypeptide comprises a Cys residue at amino acid 12 of the β2M polypeptide and (ii) the MHC class I heavy chain polypeptide comprises a Cys at residue 236, based on the numbering set forth in SEQ ID NO:19, and
wherein each variant IL-2 polypeptide has the amino acid sequence set forth in SEQ ID NO:44, wherein amino acid 16 is Ala and amino acid 42 is Ala.
17. A plurality of cells genetically modified with the nucleic acids of claim 12 .
18. A plurality of cells genetically modified with the nucleic acids of claim 16 .
19. 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.
20. 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.