Precision activation of HIV-specific CTLS to eliminate reactivated latent T cells

1. A multimeric polypeptide comprising:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) a human immune deficiency virus (HIV) epitope;

ii) a first major histocompatibility complex (MHC) polypeptide; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and

ii) optionally an immunoglobulin (Ig) Fc polypeptide or a non-Ig scaffold,

wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked; and

wherein the multimeric polypeptide comprises one or more costimulatory domains and/or cytokine(s), wherein the one or more costimulatory domain and/or cytokine is:

A) at the C-terminus of the first polypeptide;

B) at the N-terminus of the second polypeptide;

C) at the C-terminus of the second polypeptide; or

D) at the C-terminus of the first polypeptide and at the N-terminus of the second polypeptide,

and wherein the costimulatory domain is a GITRL, 4-1BBL, anti-CD28, sFv anti-CD28, CD30, LIGHT, OX40L, CD40, 4-1BBL, GITRL, ICOSL, CD70, CD80, CD86, or interferon or

wherein the costimulatory domain activates CD28, HVEM, OX40, CD40L, 4-1BB, GITR, ICOS or CD27,

or wherein the cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21 or wherein the cytokine activates a receptor for IL-2, IL-7, IL-12, IL-15 or IL-21.

2. The multimeric polypeptide of claim 1 , wherein the HIV epitope is a GAG epitope or a mutated GAG epitope.

3. The multimeric polypeptide of claim 1 , wherein the multimeric polypeptide comprises:

a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first MHC polypeptide; and

iii) one or more costimulatory domains; and

b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and

ii) an Ig Fc polypeptide.

4. The multimeric polypeptide of claim 1 , wherein the multimeric polypeptide comprises:

I) a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and

ii) a first MHC polypeptide; and

 b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) one or more costimulatory domains;

ii) a second MHC polypeptide; and

iii) an immunoglobulin (Ig) Fc polypeptide; or

II) a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and

ii) a first MHC polypeptide; and

 b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and

ii) an Ig Fc polypeptide; and

iii) one or more costimulatory domains; or

III) a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and

ii) a first WIC polypeptide; and

 b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second WIC polypeptide; and

ii) one or more costimulatory domains; or

IV) a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope; and

ii) a first WIC polypeptide; and

 b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) one or more costimulatory domains; and

ii) a second WIC polypeptide; or

V) a) a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an epitope;

ii) a first WIC polypeptide; and

iii) one or more costimulatory domains; and

 b) a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second WIC polypeptide.

5. The multimeric polypeptide of claim 1 , wherein the non-Ig scaffold is an XTEN polypeptide, a transferrin polypeptide, an Fc receptor polypeptide, an elastin-like polypeptide, a silk-like polypeptide, or a silk-elastin-like polypeptide.

6. The multimeric polypeptide of claim 1 , wherein the HIV epitope comprises the amino acid sequence SLYNTVATL, SLFNTVATL, SLFNTIATL, SLFNTVAVL, SLYNTIATL, SLYNTVAVL, SLYNTIAVL, SLFNTVATL, SLFNAVATL, SLFNAVAVL or SLFNTIAVL (SEQ ID NOS. 1-11, respectively).

7. The multimeric polypeptide of claim 1 , comprising a first linker interposed between the epitope and the first WIC polypeptide.

8. The multimeric polypeptide of claim 7 , wherein the first linker comprises a sequence -GCGAS-(GGGS) 4 - (SEQ ID NO. 14).

9. The multimeric polypeptide of claim 1 , comprising 2 or more a costimulatory domain polypeptides.

10. A nucleic acid comprising a nucleotide sequence encoding a recombinant polypeptide,

i) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus:

(1) an HIV epitope;

(2) a first major histocompatibility complex (MHC) polypeptide;

(3) one or more costimulatory domain polypeptides and/or cytokine(s);

(4) a proteolytically cleavable linker or a ribosome skipping signal;

(5) a second MHC polypeptide; and

(6) an immunoglobulin (Ig) Fc polypeptide;

wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked; or

ii) wherein the recombinant polypeptide comprises, in order from N-terminus to C-terminus:

(1) an HIV epitope;

(2) a first MHC polypeptide;

(3) a proteolytically cleavable linker or a ribosome skipping signal;

(4) one or more costimulatory domain polypeptides and/or cytokine(s);

(5) a second MHC polypeptide; and

(6) an Ig Fc polypeptide;

wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked;

and wherein the costimulatory domain is a GITRL, 4-1BBL, anti-CD28, sFv anti-CD28, CD30, LIGHT, OX40L, CD40, 4-1BBL, GITRL, ICOSL, CD70, CD80, CD86, or interferon or wherein the costimulatory domain activates CD28, HVEM, OX40, CD40L, 4-1BB, GITR, ICOS or CD27,

or wherein the cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21 or wherein the cytokine activates a receptor for IL-2, IL-7, IL-12, IL-15 or IL-21.

11. A recombinant expression vector comprising the nucleic acid of claim 10 .

12. A host cell genetically modified with the recombinant expression vector of claim 11 .

13. A composition comprising:

I) a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

i) an HIV epitope;

ii) a first MHC polypeptide; and

iii) one or more costimulatory domain polypeptides and/or cytokine(s); and

 b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) a second MHC polypeptide; and

ii) an Ig Fc polypeptide;

 wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked;

 and wherein the costimulatory domain is a GITRL, 4-1BBL, anti-CD28, sFv anti-CD28, CD30, LIGHT, OX40L, CD40, 4-1BBL, GITRL, ICOSL, CD70, CD80, CD86, or interferon or

 wherein the costimulatory domain activates CD28, HVEM, OX40, CD40L, 4-1BB, GITR, ICOS or CD27,

 or wherein the cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21 or wherein the cytokine activates a receptor for IL-2, IL-7, IL-12, IL-15 or IL-21; or

II) a) a first nucleic acid comprising a nucleotide sequence encoding a first polypeptide comprising, in order from N-terminus to C-terminus:

iii) an HIV epitope; and

iv) a first MHC polypeptide; and

 b) a first nucleic acid comprising a nucleotide sequence encoding a second polypeptide comprising, in order from N-terminus to C-terminus:

i) one or more costimulatory domain polypeptides and/or cytokine(s);

ii) a second MHC polypeptide; and

iii) an Ig Fc polypeptide;

 wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked;

and wherein the costimulatory domain is a GITRL, 4-1BBL, anti-CD28, sFv anti-CD28, CD30, LIGHT, OX40L, CD40, 4-1BBL, GITRL, ICOSL, CD70, CD80, CD86, or interferon or

wherein the costimulatory domain activates CD28, HVEM, OX40, CD40L, 4-1BB, GITR, ICOS or CD27,

or wherein the cytokine is IL-2, IL-7, IL-12, IL-15 or IL-21 or wherein the cytokine activates a receptor for IL-2, IL-7, IL-12, IL-15 or IL-21.

14. A method of producing a multimeric polypeptide, the method comprising:

a) culturing the host cell of claim 12 in vitro in a culture medium under conditions such that the host cell synthesizes the multimeric polypeptide; and

b) isolating the multimeric polypeptide from the host cell and/or from the culture medium.

15. A method of selectively modulating the activity of an HIV epitope-specific T cell, the method comprising contacting the T cell with one or more of a first multimeric polypeptide of claim 1 , wherein said contacting selectively modulates the activity of the HIV epitope-specific T cell.

16. A method of selectively modulating the activity of an HIV epitope-specific T cell in an individual, the method comprising administering to the individual an effective amount of the multimeric polypeptide of claim 1 effective to selectively modulate the activity of an HIV epitope-specific T cell in an individual.

17. A method of treating an HIV infection in an individual, the method comprising administering to the individual an amount of

a) the multimeric polypeptide of claim 1 ; or

b) one or more recombinant expression vectors comprising nucleotide sequences encoding the multimeric polypeptide of a); or

c) one or more mRNAs comprising nucleotide sequences encoding the multimeric polypeptide of a),

effective to modulate activity of HIV epitope-specific T cell in an individual.

18. A method of treating an HIV infection in an individual, the method comprising administering to the individual an amount of

a) a plurality of multimeric polypeptides of claim 1 , wherein at least two of the plurality of multimeric polypeptides comprise different HIV epitope sequences, or

b) one or more recombinant expression vectors comprising nucleotide sequences encoding a plurality of multimeric polypeptides of a), wherein at least two of the plurality of multimeric polypeptides comprise different HIV epitope sequences; or

c) one or more mRNAs comprising nucleotide sequences encoding a plurality of multimeric polypeptides of a), wherein at least two of the plurality of multimeric polypeptides comprise different HIV epitope sequences,

effective to modulate activity of HIV epitope-specific T cell in an individual.

19. A method of reactivating a latent HIV infected T-cell in an individual, the method comprising administering to the individual an amount of a multimeric polypeptide of claim 1 , wherein at least one of the first and second MHC polypeptides is HLA-mismatched with the individual, effective to reactivate a latent HIV infected T-cell in an individual.

20. A method of treating a cytomegalovirus (CMV) infection in an individual, the method comprising administering to the individual an amount of:

a) one or more multimeric polypeptides of claim 1 comprising a CMV epitope, or

b) one or more recombinant expression vectors comprising nucleotide sequences encoding a such multimeric polypeptides as described herein comprising a CMV epitope; or

c) one or more mRNAs comprising nucleotide sequences encoding multimeric polypeptides as described herein comprising a CMV epitope,

effective to modulate activity of CMV epitope-specific T cell in an individual.

21. A multimeric polypeptide of claim 1 , wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked via a disulfide bond.

22. A multimeric polypeptide of claim 10 , wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked via a disulfide bond.

23. A multimeric polypeptide of claim 13 , wherein the N-terminus of the first MHC polypeptide and the N-terminus of the second MHC polypeptide are covalently linked via a disulfide bond.

24. The method of claim 19 , wherein the at least one first and second MHC polypeptide HLA-mismatched with the individual, is allogeneic to the individual.