METHODS FOR MODULATING AN IMMUNE RESPONSE

1 . A method of modulating an immune response an individual in need thereof, the method comprising administering to the individual a multimeric polypeptide and an immune checkpoint inhibitor,

wherein the multimeric polypeptide comprises:

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

i) an 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 multimeric polypeptide comprises one or more immunomodulatory polypeptides, wherein the one or more immunomodulatory polypeptides 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 said administering modulates an immune response in the individual.

2 . The method 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) an immunomodulatory domain; 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.

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

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) an immunomodulatory domain;

iii) a second MHC polypeptide; and

ii) an immunoglobulin (Ig) Fc polypeptide.

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

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) an immunomodulatory domain.

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

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 immunomodulatory domain.

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

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) an immunomodulatory domain; and

ii) a second MHC polypeptide.

7 . The method 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) an immunomodulatory domain; and

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

i) a second MHC polypeptide.

8 . The method of claim 1 , wherein the non-Ig scaffold of the multimeric polypeptide 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.

9 . The method of any one of claims 1 - 8 , wherein the first MHC polypeptide of the multimeric polypeptide is a β2-microglobulin polypeptide; and wherein the second MHC polypeptide is an MHC class I heavy chain polypeptide.

10 . The method of claim 9 , wherein the β2-microglobulin polypeptide of the multimeric polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to one of the amino acid sequences set forth in FIG. 6 .

11 . The method of claim 9 , wherein the MHC class I heavy chain polypeptide of the multimeric polypeptide is an HLA-A, an HLA-B, or an HLA-C heavy chain.

12 . The method of claim 11 , wherein the MHC class I heavy chain polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in one of FIG. 5A-5C .

13 . The method of any one of claims 1 - 8 , wherein the first MHC polypeptide of the multimeric polypeptide is an MHC Class II alpha chain polypeptide; and wherein the second MHC polypeptide is an MHC class II beta chain polypeptide.

14 . The method of any one of claims 1 - 13 , wherein the epitope is a T-cell epitope.

15 . The method of any one of claims 1 - 7 , wherein multimeric polypeptide of the multimeric polypeptide comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

16 . The method of claim 15 , wherein the Ig Fc polypeptide comprises an amino acid sequence having at least 85% amino acid sequence identity to an amino acid sequence depicted in FIG. 4A-4C .

17 . The method of any one of claims 1 - 16 , wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are non-covalently associated.

18 . The method of any one of claims 1 - 16 , wherein the first polypeptide and the second polypeptide of the multimeric polypeptide are covalently linked.

19 . The method of claim 13 , wherein the covalent linkage is via a disulfide bond.

20 . The method of claim 19 , wherein the first MHC polypeptide or a linker between the epitope and the first MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution to provide a first Cys residue, and the second MHC polypeptide of the multimeric polypeptide comprises an amino acid substitution to provide a second Cys residue, and wherein the disulfide linkage is between the first and the second Cys residues.

21 . The method of any one of claims 1 - 8 , wherein the multimeric polypeptide comprises a linker between the epitope and the first MHC polypeptide, between the immunomodulatory polypeptide and the MHC polypeptide, or between the MHC polypeptide and the Ig Fc.

22 . The method of any one of claims 1 - 8 , wherein the immunomodulatory polypeptide of the multimeric polypeptide is selected from a 4-1BBL polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD80 polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide.

23 . The method of any one of claims 1 - 22 , wherein the multimeric polypeptide comprises 2 or more immunomodulatory polypeptides.

24 . The method of claim 23 , wherein the 2 or more immunomodulatory polypeptides are in tandem.

25 . The method of any one of claims 1 - 24 , wherein the immunomodulatory polypeptide is selected from a 4-1BBL polypeptide, a CD80 polypeptide, a CD86 polypeptide, an IL-2 polypeptide, a B7-1 polypeptide; a B7-2 polypeptide, an ICOS-L polypeptide, an OX-40L polypeptide, a CD86 polypeptide, a PD-L1 polypeptide, a FasL polypeptide, and a PD-L2 polypeptide.

26 . The method of any one of claims 1 - 25 , wherein the immunomodulatory polypeptide is a variant immunomodulatory polypeptide has one or more amino acid substitutions relative to the naturally occurring form of the immunomodulatory polypeptide, and wherein the variant immunomodulatory polypeptide exhibits reduced binding affinity to a co-modulatory polypeptide to which the naturally occurring form of the immunomodulatory polypeptide binds.

27 . The method of any one of claims 1 - 25 , wherein the immunomodulatory polypeptide is a variant IL-2 polypeptide comprising an amino acid sequence having at least 85% amino acid sequence identity to set forth in SEQ ID NO: 1,

wherein the variant IL-2 polypeptide has one or more amino acid substitutions relative to set forth in SEQ ID NO: 1, and

wherein the variant IL-2 polypeptide exhibits reduced binding affinity to an IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptides having amino acid sequences depicted in FIG. 3A-3C , compared to the binding affinity of the IL-2 amino acid sequence set forth in one of SEQ ID NO:1 for the IL2R.

28 . The method of claim 27 , wherein the variant IL-2 polypeptide comprises a substitution of one or more of E15, H16, D20, F42, Y45, and Q126.

29 . The method of claim 28 , wherein the variant IL-2 polypeptide comprises:

a) substitutions of F42 and D20;

b) substitutions of F42 and H16;

c) substitutions of F42, D20, and Y45; or

d) substitutions of F42, H16, and Q126.

30 . The method of any one of claims 1 - 21 , wherein the immunomodulatory polypeptide is a variant 4-1BBL immunomodulatory polypeptide,

wherein the variant 4-1BBL immunomodulatory polypeptide exhibits reduced binding affinity to a 4-1BB polypeptide having an amino acid sequence depicted in FIG. 37 , compared to the binding affinity of the 4-1BBL amino acid sequence depicted in FIG. 36A ; and/or

wherein the variant 4-1BBL immunomodulatory polypeptide exhibits increased production levels by a mammalian cell, compared to the production levels of the 4-1BBL amino acid sequence depicted in FIG. 36A .

31 . The method of claim 30 , wherein the variant 4-1BBL immunomodulatory polypeptide comprises a substitution of one of amino acids 91, 92, 94-115, 117-126, 128-132, 144-153, 155-158, 184-187, 189-191, 193-195, 197, 210-219, 221-224, 226, 228-231, 233, and 234 based on the amino acid numbering set out in FIG. 36A .

32 . The method of claim 30 or claim 31 , wherein the variant immunomodulatory polypeptide exhibits less than 50% of binding affinity exhibited by to the 4-1BBL amino acid sequence depicted in FIG. 36A , or as set forth in one of SEQ ID NOs:213-215, for the 4-1BB polypeptide.

33 . The method of any one of claims 1 - 32 , wherein the multimeric polypeptide comprises an Ig Fc polypeptide comprising one or more amino acid substitutions selected from N297A, L234A, L235A, L234F, L235E, and P331S.

34 . The method of claim 33 , wherein the Ig Fc polypeptide comprises:

a) an N297A substitution;

b) an L234A substitution and an L235A substitution;

c) an L234F substitution and an L235E substitution; or

d) an L234F substitution, an L235E substitution, and a P331S substitution.

35 . The method of any one of claims 1 - 34 , wherein the epitope of the multimeric polypeptide comprises the amino acid sequence YMLDLQPETT (SEQ ID NO:77).

36 . The method of any one of claims 1 - 35 , wherein the β2-microglobulin polypeptide of the multimeric polypeptide comprises the amino acid sequence depicted in FIG. 34A .

37 . The method of any one of claims 1 - 36 , wherein the major histocompatibility complex (MHC) heavy chain polypeptide of the multimeric polypeptide comprises the amino acid sequence depicted in FIG. 34C .

38 . The method of any one of claims 1 - 37 , wherein the immune checkpoint inhibitor is an antibody specific for the immune checkpoint inhibitor.

39 . The method of claim 38 , wherein the antibody is a monoclonal antibody.

40 . The method of claim 38 or claim 39 , wherein the antibody comprises at least one humanized light chain and/or heavy chain framework region.

41 . The method of claim 38 , wherein the antibody comprises an Fc polypeptide, and wherein the Ig Fc polypeptide is an IgG1 Fc polypeptide, an IgG2 Fc polypeptide, an IgG3 Fc polypeptide, an IgG4 Fc polypeptide, an IgA Fc polypeptide, or an IgM Fc polypeptide.

42 . The method of claim 38 , wherein the antibody is an Fv fragment, a nanobody, or a Fab fragment.

43 . The method of any one of claims 38 - 42 , wherein the immune checkpoint inhibitor is an antibody specific for an immune checkpoint inhibitor selected from CD27, CD28, CD40, CD122, CD96, CD73, CD47, OX40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2.

44 . The method of any one of claims 38 - 42 , wherein the immune checkpoint inhibitor is an antibody specific for PD1.

45 . The method of claim 44 , wherein the antibody is pembrolizumab, nivolumab, pidilizumab, or BMS-39886.

46 . The method of any one of claims 38 - 42 , wherein the immune checkpoint inhibitor is an antibody specific for PD-L1.

47 . The method of claim 46 , wherein the antibody is durvalumab, atezolizumab, KN035, or avelumab.

48 . The method of any one of claims 38 - 42 , wherein the immune checkpoint inhibitor is an antibody specific for CTLA4.

49 . The method of claim 48 , wherein the antibody is ipilimumab or tremelimumab.

50 . The method of any one of claims 1 - 49 , wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by the same route of administration.

51 . The method of any one of claims 1 - 49 , wherein the multimeric polypeptide and the immune checkpoint inhibitor are administered by different routes of administration.

52 . The method of any one of claims 1 - 51 , wherein the multimeric polypeptide is administered by a route of administration selected from subcutaneous, intravenous, peritumoral, and intramuscular.

53 . The method of any one of claims 1 - 51 , wherein the immune checkpoint inhibitor is administered by a route of administration selected from subcutaneous, intravenous, peritumoral, and intramuscular.

54 . The method of any one of claims 1 - 53 , wherein the individual is a human.