Immunomodulatory fusion protein-metal hydroxide complexes and methods thereof

1. An immunomodulatory fusion protein-metal hydroxide complex comprising:

(a) an immunomodulatory fusion protein comprising

(i) an immunomodulatory domain, wherein the immunomodulatory domain is or comprises a cytokine, a chemokine, an activating ligand/receptor, an inhibitory ligand/receptor, or a combination thereof, and

(ii) a metal hydroxide-binding peptide having an amino acid sequence motif comprising at least one phosphorylated amino acid, which sequence motif is or comprises:

S-X-E, S-X-pS, or S-X-Q-X-X-D-E (SEQ ID NO: 206), wherein X is any amino acid,

and

(b) a metal hydroxide

wherein the immunomodulatory fusion protein is adsorbed via ligand exchange to the metal hydroxide via the at least one phosphorylated amino acid of the metal hydroxide-binding peptide, thereby forming the immunomodulatory fusion protein-metal hydroxide complex.

2. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the immunomodulatory domain is or comprises a cytokine selected from

(i) a human gamma common chain receptor interleukin selected from IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-15/IL-15RA, IL-21 and a combination thereof,

(ii) a human IL-12 family member selected from IL-12 (p35), IL-12 (p40), IL-12(p35)/IL-12(p40), IL-23, IL-27, IL-35, and a combination thereof; and

(iii) a human IL-1 family member selected from IL-1, IL-18, IL-33, and a combination thereof; or

(iv) TNF-alpha, IFN-alpha, IFN-gamma, GM-CSF, FLT3L, G-CSF, M-CSF, and a combination thereof.

3. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the immunomodulatory domain is or comprises a chemokine selected from LIF, MIP-2, MIP-1a, MIP-1B, CXCL1, CXCL9, CXCL1O, MCP-1, Eotaxin, RANTES, LIX, CCL3, CCL4, CCL5, and a combination thereof.

4. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the immunomodulatory domain is or comprises one or more activating ligands/receptors selected from

(i) a TNF superfamily ligand selected from TNF-alpha, CD40L, 4-1BBL, OX40, or a combination thereof, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from an anti-TNFR1 antibody, an anti-TNFR2 antibody, an antiCD40 antibody, an anti-4-1BB antibody and an anti-OX40 antibody;

(ii) a CD28 superfamily member or a B7 family member selected from ICOS ligand, CD80, and CD86, or a combination thereof, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from an anti-ICOS antibody and an antiCD28 antibody; and

(iii) a T cell receptor, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from an anti-CD3γ antibody, an anti-CD3δ antibody, an anti-CD3ζ antibody, and an anti-CD3ε antibody.

5. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the immunomodulatory domain is or comprises one or more inhibitory ligands/receptors selected from

(i) a CD28 superfamily member, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment thereof selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, and an anti-CTLA4 antibody;

(ii) a TNF superfamily member, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment selected from an anti-TIGIT antibody and an anti-BTLA antibody; and

(iii) a checkpoint inhibitor, wherein the immunomodulatory domain comprises an antibody or antigen binding fragment selected from an anti-VISTA antibody, an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-CD47 antibody, and an anti-SIRPα antibody.

6. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the sequence motif is or comprises: S-X-E, wherein X is E, S, V, H, Q, or G.

7. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the metal hydroxide-binding peptide is or comprises amino acid sequence FQSEEQQ (SEQ ID NO: 129), MESEESN (SEQ ID NO:130), or GGSEEGG (SEQ ID NO: 131).

8. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the metal hydroxide-binding peptide is or comprises amino acid sequence GGSEEGG (SEQ ID NO: 131).

9. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the metal hydroxide-binding peptide is operably linked to either the N-terminus or C-terminus of the immunomodulatory domain.

10. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the metal hydroxide is selected from aluminum hydroxide, aluminum phosphate, calcium hydroxide, calcium phosphate, iron hydroxide, magnesium hydroxide, barium hydroxide, calcium hydroxide, zinc hydroxide, and zirconium hydroxide.

11. The immunomodulatory fusion protein-metal hydroxide complex of claim 1 , wherein the metal hydroxide is aluminum hydroxide.