ZARBIS-PAPASTOITSIS GRIGORIOS (US)
WITTRUP KARL DANE (US)
IRVINE DARRELL (US)
WO2020263399A1 | 2020-12-30 |
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Claims We claim: 1. A fusion polypeptide comprising: (a) an immunomodulatory polypeptide that comprises an immune agonist moiety; and (b) a metal-hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms. 2. The fusion polypeptide of claim 1, wherein the fusion polypeptide, when exposed to a metal-hydroxide forms a complex therewith. 3. The complex of claim 2, wherein the metal hydroxide is aluminum hydroxide. 4. The complex of claim 2, wherein the complex forms more readily when the metal- hydroxide-binding polypeptide is in a phosphorylated form than when it is in an unphosphorylated form. 5. The complex of claim 4, wherein one or more of the phosphorylation sites is targeted by a Fam20C kinase. 6. The complex of claim 5, wherein the phosphorylation site is or comprises an S-X-E motif. 7. The fusion polypeptide of claim 1, wherein the plurality of phosphorylation sites comprises more than 4 S-X-E motifs. 8. The fusion polypeptide of claim 7, wherein the plurality of phosphorylation sites comprises 8 S-X-E motifs. 9. The fusion polypeptide of claim 7, wherein at least two adjacent S-X-E motifs are separated by a spacer. 10. The fusion polypeptide of claim 9, wherein the spacer comprises at least one glycine residue. 11. The fusion polypeptide of claim 10, wherein the spacer comprises a plurality of glycine residues. 12. The fusion polypeptide of claim 11, wherein the spacer comprises at least four glycine residues. 13. The fusion polypeptide of claim 12, wherein the spacer has a sequence that comprises four glycine residues. 14. The fusion polypeptide of claim 7, wherein each SXE motif is separated from each adjacent S-X-E motif by a spacer. 15. A method of treating a subject with a tumor, the method comprising a step of: treating the subject with a complex comprising: (a) fusion polypeptide comprising: (i) an immunomodulatory polypeptide that comprises an immune agonist moiety; and (ii) a metal-hydroxide binding peptide; and, (b) a metal hydroxide. 16. The method of claim 15, wherein (a) and (b) are formulated together. 17. The method of claim 15, wherein (a) and (b) are mixed prior to administration. 18. The method of claim 15, wherein the complex is administered by intratumoral injection. 19. The method of claim 15, wherein the complex is administered by peritumoral injection. 20. The method of claim 15, wherein the complex is administered to a tumor-draining lymph node or lymph nodes. 21. The method of claim 15, wherein the complex is administered in combination with a second therapeutic. 22. The method of claim 21, wherein the second therapeutic is radiation. 23 The method of claim 21, wherein the second therapeutic is surgical tumor resection. 24. The method of claim 23, wherein the fusion polypeptide is administered prior to surgical tumor resection. 25. The method of claim 21, wherein the second therapeutic is a chemotherapy or targeted therapy. 26. The method of claim 21, wherein the second therapeutic is an anti-tumor antibody. 27. The method of claim 21, wherein the second therapeutic is an immune modulator. 28. The method of claim 27, wherein the immune modulator is a checkpoint inhibitor. 29. The method of claim 28, wherein the checkpoint inhibitor is an antibody or a functional fragment thereof. 30. The method of claim 29, wherein the antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and LAG3. 31. The method of claim 30, wherein the antibody targets PD-1. 32. The method of claim 26, wherein the antibody is a tumor-targeting CD3 bispecific antibody. 33. The method of claim 27, wherein the immune modulator is a cell therapy. 34. The method of claim 33, wherein the cell therapy is selected from the group consisting of: CAR-T cells, ex-vivo expanded TILs, and NK cells. 35. A method of treating a subject with a tumor comprises administering a fusion polypeptide comprising: (a) an immunomodulatory polypeptide that comprises an immune agonist moiety; and (b) a metal-hydroxide binding peptide, wherein the fusion polypeptide is formulated with a metal hydroxide; and wherein the subject has received or is receiving therapy with at least one additional therapeutic. 36. The method of claim 35, wherein the fusion polypeptide and metal-hydroxide are formulated together, forming a complex. 37. The method of claim 35, wherein the fusion polypeptide and metal-hydroxide are mixed prior to administration. 38. The method of claim 35, wherein the fusion polypeptide is administered by intratumoral injection. 39. The method of claim 35, wherein the fusion polypeptide is administered by peritumoral injection. 40. The method of claim 35, wherein the fusion polypeptide is administered to a tumor- draining lymph node or lymph nodes. 41. The method of claim 35, wherein the at least one additional therapeutic is radiation. 42. The method of claim 35, wherein the at least one additional therapeutic is surgical tumor resection. 43. The method of claim 42, wherein the fusion polypeptide is administered prior to surgical tumor resection. 44. The method of claim 35, wherein the at least one additional therapeutic is a chemotherapy or a targeted therapy. 45. The method of claim 35, wherein the at least one additional therapeutic is an anti-tumor antibody. 46. The method of claim 45, wherein the second therapeutic is an immune modulator. 47. The method of claim 46, wherein the immune modulator is a checkpoint inhibitor. 48. The method of claim 47, wherein the checkpoint inhibitor is an antibody or a functional fragment thereof. 49. The method of claim 48, wherein the antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and LAG3. 50. The method of claim 49, wherein the antibody targets PD-1. 51. The method of claim 45, wherein the antibody is a tumor-targeting CD3 bispecific antibody. 52. The method of claim 46, wherein the immune modulator is a cell therapy. 53. The method of claim 52, wherein the cell therapy is selected from the group consisting of: CAR-T cells, ex-vivo expanded TILs, NK cells, and macrophages. 54. The fusion polypeptide of claim 1, wherein the immune agonist moiety comprises a first moiety or a functional fragment thereof. 55. The fusion polypeptide of claim 54, wherein the functional fragment is signaling competent. 56. The fusion polypeptide of claim 54, wherein the first moiety comprises an IL12 moiety or a functional fragment thereof. 57. The fusion polypeptide of claim 56, wherein the IL12 moiety comprises IL12B or a functional fragment thereof. 58. The fusion polypeptide of claim 1, wherein the immune agonist moiety comprises a first and a second moiety or a functional fragment thereof. 59. The fusion polypeptide of claim 58, wherein the first moiety comprises an IL12 moiety or a functional fragment thereof. 60. The fusion polypeptide of claim 59, wherein the IL12 moiety comprises IL12B or a functional fragment thereof. 61. The fusion polypeptide of claim 58, wherein the second moiety comprises an IL12 moiety or a functional fragment thereof. 62. The fusion polypeptide of claim 61, wherein the second IL12 moiety comprises IL12A or a functional fragment thereof. 63. The fusion polypeptide of claim 58, wherein the first and second moieties or functional fragments thereof are linked via a first linker. 64. The fusion polypeptide of claim 63, wherein the first linker comprises a polypeptide. 65. The fusion polypeptide of claim 64, wherein the polypeptide comprises a (G4S)3 linker. 66. The fusion polypeptide of claim 1, wherein an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide are linked via a second linker. 67. The fusion polypeptide of claim 66, wherein the second linker comprises a polypeptide. 68. The fusion polypeptide of claim 67, wherein the polypeptide comprises the amino acid sequence, GGGGEGGGG. 69. The fusion polypeptide of claim 67, wherein the polypeptide comprises the amino acid sequence, GGGGSGGGG. 70. The fusion polypeptide of claim 1, wherein the metal-hydroxide binding polypeptide is linked directly to the c-terminus of the immunomodulatory polypeptide. 71. The fusion polypeptide of claim 1, wherein the metal-hydroxide binding polypeptide is linked via a second linker to the c-terminus of the immunomodulatory polypeptide. 72. A method of manufacturing a phosphorylated form of the fusion polypeptide of claim 1, by contacting the fusion polypeptide with a kinase. 73. The method of claim 72, wherein the contacting comprises co-expressing the fusion polypeptide and a kinase. 74. The method of claim 73, wherein the fusion polypeptide and kinase are co-expressed at a ratio of 2:1 to 100:1. 75. The method of claim 74, wherein the ratio is 4:1. 76. The method of claim 75, wherein the 4:1 ratio is achieved using two separate plasmids to express the fusion polypeptide and the kinase. 77. The method of claim 76, wherein the two separate plasmids comprise promoters of differing strength to produce the ratio of 4:1. 78. The method of claim 74, wherein the ratio is 8:1. 79. The method of claim 78, wherein the 8:1 ratio is achieved using a single vector with two promoters to express the fusion polypeptide and the kinase. 80. The method of claim 72, wherein the kinase is Fam20C. 81. The method of claim 72, the method further comprising a step of purifying the phosphorylated form. 82. The method of claim 81, wherein the step of purifying comprises chromatography. 83. The method of claim 73, wherein the step of co-expressing comprises expressing from promoters established to direct expression at the ratio. 84. The method of claim 73, where the step of co-expressing comprises expressing from a bi- cistronic construct. 85. The method of claim 72, wherein the fusion polypeptide is exposed to a metal-hydroxide to form a complex therewith. 86. The method of claim 85, wherein the complex is prepared prior to administering to a subject. 87. The method of manufacturing a complex by contacting a phosphorylated form of a fusion polypeptide of claim 1 with a metal hydroxide. 88. A complex comprising the fusion polypeptide of claim 1 and a metal hydroxide. 89. The complex of claim 88, wherein the complex comprises an average of 2-8 phosphates per fusion polypeptide. 90. The complex of claim 88, wherein the complex is characterized as having greater than 95% metal hydroxide retention. 91. The complex of claim 88, wherein the complex comprises a ratio of 1:1 to 1:20 by mass of fusion polypeptide to metal hydroxide as defined by metal mass. 92. The complex of claim 91, wherein the ratio is 1:5 to 1:20 by mass of fusion polypeptide to metal hydroxide as defined by metal mass. 93. The complex of claim 91, wherein the ratio is 1:10 by mass of fusion polypeptide to metal hydroxide as defined by metal mass. 94. The complex of claim 91, wherein the ratio is 1:5 by mass of fusion polypeptide to metal hydroxide as defined by metal mass. 95. A pharmaceutical composition comprising the fusion polypeptide of claim 1. 96. The pharmaceutical composition of claim 95, formulated as a fusion polypeptide metal- hydroxide complex. 97. The method of characterizing a preparation of a fusion polypeptide of claim 1 by assessing degree of phosphorylation. 98. The method of claim 97, wherein the degree of phosphorylation is assessed by determining the number of phosphates per protein. 99. The method of claim 98, wherein the number of phosphates per protein is determined using a colorimetric assay. 100. The method of claim 99, wherein the colorimetric assay is a malachite green assay. 101. The method of characterizing a preparation of a fusion polypeptide of claim 1 by assessing heterogeneity of the preparation. 102. The method of claim 101, wherein heterogeneity of the preparation is assessed using analytical ion exchange. 103. The method of characterizing a preparation of a fusion polypeptide of claim 1 by assessing retention of the fusion polypeptide on the metal hydroxide. 104. The method of claim 103, wherein retention is assessed using an in vitro retention assay. 105. A method of characterizing a preparation of a fusion polypeptide of claim 1 by assessing signaling activity of the fusion polypeptide. 106. The method of claim 105, wherein the signaling activity is assessed in vitro. 107. The method of claim 106, wherein in vitro assessment utilizes a reporter assay. 108. The method of characterizing a preparation of a fusion polypeptide of claim 1 comprising assessing purity of the preparation. 109. The method of characterizing a preparation of a fusion polypeptide of claim 1 comprising assessing phosphate content. 110. The method of claim 109, wherein assessing phosphate content comprises use of a malachite green assay. 111. The method of claim 110, wherein assessing phosphate content comprises use of a high performance liquid chromatography assay. 112. The method of claim 111, wherein the high performance liquid chromatography assay utilizes a SAX-10 column. 113. The method of characterizing a preparation of a fusion polypeptide of claim 1 comprising assessing potency of the preparation. 114. The method of claim 113, wherein potency is characterized by assessing immune moiety signaling. 115. The method of claim 114, wherein immune moiety signaling is determined using a reporter assay. 116. The method of claim 114, wherein potency is characterized by assessing IL12 signaling. 117. The method of claim 116, wherein IL12 signaling is determined using a reporter assay. 118. A method of characterizing the complex of claim 88 comprising assessing retention of the fusion polypeptide to the metal hydroxide. 119. The method of claim 118, wherein assessing retention comprises use of a metal hydroxide retention assay. 120. A method of characterizing a pharmaceutical composition of claim 95 comprising assessing one or more of: (a) the purity of the preparation; (b) phosphate content; (c) potency of the pharmaceutical composition; (d) retention of the fusion polypeptide to the metal hydroxide; (e) efficacy of treating a subject having a tumor; and (f) combination with a second therapeutic agent. |
[0129] Table 2 provides exemplary nucleotide sequences encoding polypeptides described herein. Table 2: Exemplary Nucleotide Sequences Fusion polypeptides Immunomodulatory polypeptide [0130] Fusion polypeptides of the present disclosure comprise at least one immunomodulatory polypeptide. [0131] In some embodiments, an immunomodulatory polypeptide is or comprises at least one immune agonist moiety. In some embodiments, an immune agonist moiety is or comprises a functional fragment of a parent (e.g., a wild type) polypeptide; for example, in some embodiments, an immunomodulatory polypeptide is or comprises a functional fragment that is a signaling competent fragment. In some embodiments, an immunomodulatory polypeptide comprises one, two, three, four, five, or six immune agonist moieties. [0132] In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties). In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that are the same; in some such embodiments, all immunomodulatory polypeptides in a fusion polypeptide in accordance with the present disclosure are the same. In some such embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides that at are different from one another. [0133] Thus, in some embodiments, an immunomodulatory polypeptide may include more than one immune agonist moiety which, in various embodiments, may be the same or different. In some such embodiments, two or more such immune agonist moieties are the same; in some embodiments, all such immune agonist moieties are the same. In some embodiments, an immunomodulatory polypeptide includes two or more immune agonist moieties that differ from one another; in some embodiments, no two such immune agonist moieties are the same. [0134] In some embodiments, a fusion polypeptide comprises two or more immunomodulatory polypeptides (e.g., two or more immune agonist moieties) that include at least two subtypes of immunomodulatory polypeptides (e.g., immune agonist moieties) – for example so that the fusion polypeptide includes at least two of a first subtype and at least two of a second subtype. In some such embodiments, a subset of the same immunomodulatory polypeptides (e.g., immune agonist moieties) equals 1 immunomodulatory polypeptide out of 2 total moieties in the fusion polypeptide, 2 immunomodulatory polypeptides out of 2 total, 1 immunomodulatory polypeptides out of 3, 2 immunomodulatory polypeptides out of 3 total, or 3 immunomodulatory polypeptides out of 3. In some such embodiments, a subset of different immunomodulatory polypeptides equals 1 immune immunomodulatory polypeptides out of 2 total, 2 immunomodulatory polypeptides out of 2 total, 1 immunomodulatory polypeptides out of 3 total, 2 immunomodulatory polypeptides out of 3 total, or 3 immunomodulatory polypeptides out of 3 total. [0135] In some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) activates or inhibits activity of a cell of the immune system (e.g., is signaling competent). In some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) is assessed, for example as part of a fusion polypeptide, e.g., as described herein. [0136] For example, in some embodiments, signal competency is characterized in that, when assessed for binding to a particular binding partner, an immune agonist moiety or moieties or functional fragments thereof displays binding comparable to that of a reference standard (e.g., a wild-type polypeptide). For example, in some embodiments, signal competency is characterized in that, when assessed for a biological effect, e.g., in vitro or in vivo, an immune agonist moiety or moieties or functional fragments thereof displays said biological effect comparable to that of a reference standard (e.g., a wild-type polypeptide). [0137] For example, in some embodiments, an immunomodulatory polypeptide (e.g., an immune agonist moiety) is an immune response stimulatory moiety. In some embodiments, a response stimulatory moiety is, for example, but without limitation, a cytokine, a chemokine, an agonistic antibody, an immune checkpoint inhibitor, or a combination thereof. IL-12 [0138] In some embodiments, an immunomodulatory polypeptide comprises an interleukin-12 (IL-12) immunomodulatory polypeptide (e.g., an IL-12 immune agonist moiety). [0139] IL-12 is a pro-inflammatory cytokine that plays an important role in innate and adaptive immunity. Wild type IL-12 is a heterodimeric protein comprising two subunits, p35 (IL-12A; GenBenk GeneID: 3592) and p40 (IL-12B; GenBank GeneID: 3593), connected by disulfide bonds. Binding of IL-12 to the IL-12 receptor complex (IL-12Rβ1 / IL-12Rβ2) on T cells and Natural Killer (NK) cells leads to signaling via signal transducer and activator of transcription 4 (STAT4) and subsequent interferon γ (IFN-γ) production and secretion. [0140] IL-12 subunits, IL-12A and IL-12B, can also form heterodimers with other IL-12 family members. For example, IL-12A may also dimerize with Epstein-Barr virus induced gene 3 (EBI3) to form IL-12 family member, IL-35 and IL-12B may dimerize with a p19 monomer, to form IL-12 family member, IL23. [0141] IL-12 plays important roles in the innate and adaptive immune response, and dysregulation of IL-12 has been implicated in a number of disease states. Exemplary such disease states include, but are not limited to, inflammatory bowel disease, psoriasis, diabetes mellitus, multiple sclerosis, rheumatoid arthritis, cancer, lupus erythematosus, primarily biliary cholangitis and Sjögren's syndrome (Ullrich et al. EXCLI journal vol.191563-1589. 11 Dec.2020). Use of IL-12 as a therapeutic modality has been studied extensively, including for treatment of tumors (Nastala CL et al. J Immunol.1994 Aug 15; Lasek et al. Cancer immunology, immunotherapy: CII vol.63,5 (2014): 419-35). [0142] In some embodiments, an immunomodulatory polypeptide disclosed herein is or comprises an IL-12 immune agonist moiety. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises a plurality of IL-12 immune agonist moieties. In some embodiments, an immunomodulatory polypeptide disclosed herein comprises exactly two IL-12 immune agonist moieties. In some embodiments, two or more IL-12 moieties of a plurality of (e.g., two) IL-12 moieties are the same moiety. In some such embodiments, a plurality (e.g., two) IL-12 moieties are different moieties. In some such embodiments, an IL-12 moiety comprises an IL-12A polypeptide or functional fragment thereof. In some embodiments, an IL-12 moiety comprises an IL-12B polypeptide or functional fragment thereof. [0143] In some embodiments, an IL-12B immune agonist moiety is located N- terminal to an IL-12A immune agonist moiety in an immunomodulatory polypeptide. In some embodiments, an IL-12A immune agonist moiety is located N-terminal to an IL-12B immune agonist moiety in an immunomodulatory polypeptide. [0144] In some embodiments, an immunomodulatory polypeptide comprising a plurality (e.g., two) IL-12 moieties (e.g., IL-12A and/or IL-12B) are linked directly. In some embodiments, an immunomodulatory polypeptide comprising a plurality (e.g., two) IL-12 moieties (e.g., IL-12A and/or IL-12B) are linked via a first linker. Non-limiting examples of linkers are discussed elsewhere herein. [0145] In some embodiments, an immunomodulatory polypeptide disclosed herein comprises an IL-12A and/or IL-12B immune agonist moiety comprising a variant. In some embodiments, an IL-12A and/or IL-12B immune agonist moiety variant comprises a substitution, deletion, addition, and/or insertion of relative to a wild-type IL-12A or IL-12B polynucleotide or amino acid sequence. In some embodiments, an IL-12A and/or IL-12B immune agonist moiety comprises a plurality of variants. In some embodiments, a plurality of variants comprises one or more of a substitution, deletion, addition, and/ or insertion relative to a wild-type IL-12A or IL-12B. In some embodiments, a variant comprises a substitute that does not change the amino acid sequence relative to a wild-type IL-12A or IL-12B. [0146] In some embodiments, an IL12 variant comprises a S43X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S154X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S168X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S227X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S233X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a T364X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than T. In some embodiments, an IL12 variant comprises a S365X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S398X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S365X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S480X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S481X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S365X and S481X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises a S365X, S398X, and S481X mutation relative to SEQ ID NO: 25, wherein X is any amino acid other than S. In some embodiments, an IL12 variant comprises any combination of variants, including, for example, those disclosed herein. [0147] In some embodiments, an immunomodulatory polypeptide disclosed herein comprises an IL-12A and/or IL-12B immune agonist moiety that is a functional fragment thereof (e.g., a signaling competent fragment). In some embodiments, an immunomodulatory polypeptide comprises a functional IL-12A fragment. In some embodiments, an immunomodulatory polypeptide comprises a functional IL-12B fragment. In some embodiments, an immunomodulatory polypeptide comprises a full length IL-12A and a functional IL-12B fragment. In some embodiments, an immunomodulatory polypeptide comprises a full length IL-12B and a functional IL-12A fragment. [0148] In some embodiments, a IL-12A or IL-12B fragment comprises or consists of at least 5%, 10,%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the monomeric units (e.g., residues) as found in wild-type IL-12A or IL-12B. Metal-hydroxide binding polypeptide [0149] The Fusion Protein Filing has taught that hydroxyl replacement (e.g., with phosphate groups) can increase a polypeptide’s adsorption via ligand exchange with a metal hydroxide (e.g., aluminum hydroxide), and furthermore can improve tumor retention and anti-tumor efficacy of such polypeptide (e.g., specifically of a fusion polypeptide comprising an immunomodulatory polypeptide and a metal-hydroxide-binding polypeptide in which such hydroxyl replacement has occurred. [0150] As discussed above, metal-hydroxide- (e.g., alum-) binding polypeptides were developed that could be fused to an immunomodulatory polypeptide to allow strong binding to aluminum hydroxide. Various immunomodulatory polypeptides fused to ABPs were assessed. It was determined that the ABPs adsorbed to alum in serum and could be used to retain proteins and peptides in tumors. Additionally, polypeptides with greater phosphorylation tended to be retained on alum for much longer in serum conditions. Of the ABPs analyzed, the polypeptide, referred to as ABP10, demonstrated the highest phosphorylation with an increase of phosphorylation of 4-6-fold when the protein was expressed with a wild-type (WT) kinase (e.g., WT Fam20C kinase), compared to when the protein was expressed with a mutant kinase (e.g., mutant Fam20C kinase). ABP10 consists of four SXE motifs, a prevalent phosphorylation site motif, separated by short spacer sequences. It was demonstrated immunomodulatory polypeptides (e.g., interleukin-2 and interleukin-12) linked to ABP10 showed improved survival in a mouse model of melanoma compared to the immunomodulatory polypeptides without ABP10. [0151] We have surprisingly found that improved metal-hydroxide binding polypeptides can be developed. Among other things, we have developed metal-hydroxide binding polypeptides characterized by enhanced metal hydroxide (e.g., alum) retention relative to an appropriate reference (e.g., to ABP10). Alternatively or additionally, provided metal-hydroxide binding polypeptides are characterized by improved efficacy, as compared to an appropriate reference (e.g., to ABP10), when administered to a subject with a tumor. [0152] Thus, among other things, the present disclosure identifies the source of a problem with certain metal-hydroxide binding polypeptides and/or fusion polypeptides that include them. For example, the present disclosure appreciates that manufacturing challenges can be associated with certain such polypeptides and/or fusions. Without wishing to be bound by any particular theory, the present disclosure notes that secondary phosphorylation on the polypeptide may contribute to and/or be responsible for certain such manufacturing challenges. Among other things, the present disclosure provides metal-hydroxide-binding polypeptides, and fusion polypeptides that include them, which demonstrate high levels of adsorption to metal hydroxides and also desirable manufacturing characteristics (e.g., one or more of reproducibility, consistency, production of a homogeneously-phosphorylated preparation, etc.). [0153] In some embodiments, a metal-hydroxide binding polypeptide comprises an amino acid sequence that includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms. In some embodiments, a metal-hydroxide binding polypeptide comprises at least one kinase target motif. A target kinase motif comprises an amino acid that is phosphorylated by a kinase. Amino acids that are typically phosphorylated include a hydroxyl, such as serine (Ser, S), threonine (Thr, T), and tyrosine (Tyr, Y) residues. A kinase motif refers to the amino acid sequence immediately N- and/or C-terminal to the amino acid residue capable of being phosphorylated. Without wishing to be bound by any one theory, many kinases comprise structural features that confer specificity such that the kinase phosphorylates a particular amino acid (e.g., serine, threonine, or tyrosine) of a particular kinase target motif. [0154] Kinase target motifs recognized are highly diverse depending on the particular type of kinase. In some embodiments, the present disclosure provides metal- hydroxide binding polypeptides comprising one or more kinase target motifs of a secretory pathway kinase. The secretory pathway, which is the pathway by which a cell secretes proteins and/or other biomolecules into the extracellular space, refers to the endoplasmic reticulum (ER), Golgi apparatus (Golgi), cell membrane, and lysosomal storage compartments as well as the vesicles that travel between them. Secretory pathway kinases are localized throughout the secretory pathway (e.g., at the ER, Golgi, etc.) and function to phosphorylate proteins destined for secretion (Sreelatha et al. Biochimica et biophysica acta vol.1854,10 Pt B (2015): 1687-93). [0155] In some embodiments, a relevant kinase is a naturally occurring secretory pathway kinase (e.g., is endogenously targeted to the secretory pathway to function). In some embodiments, a secretory pathway kinase comprises a signal sequence that targets the kinase to the secretory pathway. Naturally-occurring human secretory pathway kinases include, for example, four-jointed box kinase 1, Fam20A, Fam20B, Fam20C, vertebrate lonesome kinase (VLK), SGK196, and Fam69A, Fam69B, and Fam69C. [0156] In some embodiments, a relevant kinase is a non-naturally occurring secretory pathway kinase. In some embodiments, a non-naturally occurring kinase is produced by linking a secretory signal peptide to a kinase endogenously localized to a non- secretory pathway cellular compartment. [0157] In some embodiments, a kinase target motif is a target kinase motif of a secretory pathway kinase. In some embodiments, a secretory pathway kinase target kinase motif comprises an S-X-E motif. For example, Fam20C phosphorylates serine and has been shown to phosphorylate kinase targets motif comprising the amino acid sequence Ser-X-Glu (e.g., S-X-E), Ser-X-pSer (e.g., S-X-pS), and Ser-X-Gln-X-X-Asp-Glu-Glu (S-X-Q-X-X-D- E-E) wherein X is any amino acid, and pS is phosphorylated serine (Mercier, et al (1981) Biochimie, 63: 1-17; Mercier et al (1971) Eur J. Biochem.23:41-51; Lasa-Benito (1996) FEES Lett.382:149; Brunati, et al (2000) 3:765, Tagliabracci, et al (2015) Cell 161:1619- 1632; Tagliabracci, et al (2012) Science 336:1150-1153). In some embodiments, a target kinase motif comprises the amino acid sequence SEEE. In some embodiments, a target kinase motif comprises the amino acid sequence SEEA. In some embodiments, a target kinase motif comprises the amino acid sequence SEEQ. In some embodiments, a target kinase motif comprises the amino acid sequence SEE. [0158] In some embodiments, a metal-hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises more than four S-X-E motifs. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X- E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide. [0159] In some embodiments, a metal-hydroxide binding polypeptide is a metal- hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites. In some embodiments, a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs. In some embodiments, a plurality of phosphorylation sites comprises at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than four S-X-E motifs. In some embodiments, a plurality of phosphorylation sites comprises more than eight S-X-E motifs. In some embodiments, the number of target kinase motifs (e.g., S-X-E motifs) contributes to the number of phosphorylated residues on a metal-hydroxide binding polypeptide. [0160] In some embodiments, the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (e.g., S-X-E motifs) are directly adjacent (e.g., linked) to the next target kinase (e.g., S-X-E motif). In some embodiments, the at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve target kinase motifs (e.g., S-X-E motifs) are separated (e.g., linked) to the next target kinase motif (e.g., S-X-E motif) by a spacer. In some embodiments, the spacer comprises at least one glycine residue. In some embodiments, the spacer comprises a plurality of glycine residues. In some embodiments, the spacer comprises three glycine residues. In some embodiments, the spacer comprises at least four glycine residues. In some embodiments, the spacer has a sequence that comprises four glycine residues. In some embodiments, the spacer has an amino acid sequence comprising GGGSGGGG. In some embodiments, the spacer has an amino acid sequence comprising GGGEGGGG. [0161] In some embodiments, a metal-hydroxide binding polypeptide comprises four S-X-E motifs and three spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises six S-X-E motifs and five spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X-E motifs and seven spacers comprising four glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEE, and three spacers comprising three glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEA, and three spacers comprising three glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEEQ, and three spacers comprising three glycine residues. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEE, and three spacers comprising the amino acid sequence, GGGSGGGG. In some embodiments, a metal-hydroxide binding polypeptide comprises four motifs with the amino acid sequence, SEE, and three spacers comprising the amino acid sequence, GGGEGGGG. [0162] In some embodiments, a metal-hydroxide binding polypeptide comprises six S-X-E motifs, wherein each S-X-E motif is directly adjacent to the next S-X-E motif. In some embodiments, a metal-hydroxide binding polypeptide comprises eight S-X-E motifs, wherein each S-X-E motif is directly adjacent to the next S-X-E motif. [0163] In some embodiments, a metal-hydroxide binding polypeptide comprise an ending sequence (e.g., an amino acid sequence at the c-terminus of the fusion polypeptide. In some embodiments, an ending sequence comprises a plurality of amino acid residues. In some embodiments, a plurality of amino acid residues comprises GGGG. In some such embodiments, an ending sequence comprises the amino acid sequence GGGGS. In some such embodiments, an ending sequence comprises the amino acid sequence GGGGHHHHHH In some such embodiments, an ending sequence comprises the amino acid sequence GGGGSHHHHHH.In some embodiments, an ending sequence comprises an optional tag, as discussed elsewhere herein. [0164] Among other things, the present disclosure identifies the source of a problem with certain metal-hydroxide binding polypeptides and/or fusion polypeptides that include them. Among other things, the present disclosure provides metal-hydroxide-binding polypeptides, and fusion polypeptides that include them, which demonstrate high levels of adsorption to metal hydroxides and also desirable manufacturing characteristics (e.g., one or more of reproducibility, consistency, production of a homogeneously-phosphorylated fusion polypeptide, etc.). For example, in some embodiments, an optimal number of kinase target motifs (e.g., phosphorylation sites) are utilized. In some embodiments, for example, spacing of kinase target motifs (e.g., phosphorylation sites) is or has been optimized. [0165] In some embodiments, a desired (e.g., optimal) number of kinase target motifs and/or spacing of kinase motifs may be determined based on one or more of, for example, desired phosphate content to achieve strong metal-hydroxide retention and/or avoidance of one or more manufacturing challenges (e.g., which the present disclosure appreciates may be associated with highly phosphorylated elements). In some embodiments, a desired (e.g., optimal) number and/or spacing of kinase motifs results in an exposure of the polypeptide to the kinase to achieve a desired level of fusion polypeptide phosphorylation. In some embodiments, improved fusion polypeptides as described herein, results in one or more of improved reproducibility, consistency, and/or production of a homogenously- phosphorylated fusion polypeptide. For example, in some embodiments, provided technologies achieve reproducible manufacturing of comparable preparations (e.g., preparations that are consistently within established parameters) of fusion polypeptides (e.g., phosphorylated fusion polypeptides) and/or complexes as described herein. For example, in some embodiments, provided technologies achieve reduced immunogenicity compared to an appropriate reference standard (e.g., fusion polypeptides comprising ABP-10). Without wishing to be bound by any one theory, reduced immunogenicity is achieved by removing hydrophobic amino acids, thus, reducing binding to major histocompatibility complex. Linkers [0166] In some embodiments, fusion polypeptides as described herein may include one or more linkers or spacers. [0167] For example, in some embodiments, fusion polypeptides comprise an immunomodulatory polypeptide comprising a first and a second immune agonist moiety. In some embodiments, a first and a second immune agonist moiety are linked via a first linker. [0168] In some embodiments, fusion polypeptides of the present disclosure comprise an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide. In some embodiments, an immunomodulatory polypeptide and a metal-hydroxide binding polypeptide are linked via a second linker. [0169] In some embodiments, a first linker and/or a second linker is a polypeptide linker. In some embodiments, a polypeptide linker is synthetic. For example, a synthetic polypeptide linker may comprise non-naturally occurring polypeptides which are modified forms of naturally occurring polypeptides. [0170] In some embodiments, polypeptide linkers of the present disclosure are at least one amino acid in length and can be any suitable number of amino acids. In some embodiments, a polypeptide linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. [0171] In some embodiments, a first linker comprises a polypeptide linker. In some embodiments, a first linker comprises or consists of a Glycine-Serine (Gly-Ser or G-S linker). A Gly-Ser linker is a polypeptide linker that consists of glycine and serine residues. In some embodiments, a Gly-Ser linker comprises an amino acid sequence of (Gly4Ser)n, wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, a Gly-Ser linker is (Gly4Ser)1. In some embodiments, a Gly-Ser linker is (Gly4Ser)2. In some embodiments, a Gly-Ser linker is (Gly4Ser)3.. In some embodiments, a Gly-Ser linker is (Gly 4 Ser) 4. In some embodiments, a Gly-Ser linker is (Gly 4 Ser) 5. In some embodiments, a Gly-Ser linker is (Gly4Ser)6. In some embodiments, a Gly-Ser linker is (Gly4Ser)7. In some embodiments, a Gly-Ser linker is (Gly 4 Ser) 8. In some embodiments, a Gly-Ser linker is (Gly4Ser)9. In some embodiments, a Gly-Ser linker is (Gly4Ser)10. [0172] In some embodiments, a second linker comprises a polypeptide linker. In some embodiments, a second linker comprises a plurality of glycine residues. In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGSGGGG. In some embodiments, a second linker comprises a polypeptide linker with the amino acid sequence, GGGGEGGGG. Variants [0173] In some embodiments, an immunomodulatory polypeptide or a metal- hydroxide-binding polypeptide utilized in accordance with the present disclosure is a variant of a relevant reference polypeptide (e.g., a wild type polypeptide or functional portion thereof). [0174] In some embodiments, a variant shows at least 70% identity to its reference polypeptide. In some such embodiments, a variant shows at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to its references polypeptide [0175] In some embodiments, a variant comprises one or more conservative or otherwise non-disruptive modifications (e.g., substitutions, deletions or additions) relative to its reference. In some embodiments, a variant is free of any disruptive modifications (e.g., substitutions, deletions or additions) so that an immunomodulatory polypeptide maintains one or more functional characteristics of the reference. In some embodiments, maintains means an immunomodulatory polypeptide display comparable activity (e.g., signaling competency or binding) compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). For example, in some such embodiments, an immunomodulatory polypeptide maintains at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher activity compared to an appropriate reference standard (e.g., a wild-type immunomodulatory polypeptide). Metal hydroxides [0176] In some embodiments, the present disclosure provides fusion polypeptides comprising an immunomodulatory polypeptide and a metal-hydroxide binding polypeptides, wherein the fusion polypeptide, when exposed to a metal-hydroxide forms a complex therewith. A complex is formed via adsorption of the fusion polypeptide to a metal- hydroxide. Without wishing to be bound by any one theory, it is hypothesized adsorption of a fusion polypeptide to a metal hydroxide occurs by ligand exchange. Ligand exchange, for example, is a substitution or exchange of a surface hydroxyl by another ligand. In some embodiments, substitution or exchange of a surface hydroxyl occurs by a hydroxyl- replacement group (e.g., a phosphate group). [0177] In some embodiments, a metal-hydroxide is a substance that includes at least one hydroxyl group bound to a metal. In accordance with the present disclosure, in some embodiments, a metal-hydroxide can adsorb fusion polypeptides comprising a hydroxyl- replacement moiety. In some embodiments, a hydroxyl-replacement moiety is a phosphate group. [0178] In some embodiments, a metal-hydroxide is selected based on its inherent qualities or characteristics. In some embodiments, a metal-hydroxide is selected due to its biocompatibility for use in a subject (e.g., a mammal, e.g., a human). In some embodiments, a metal-hydroxide is aluminum hydroxide (e.g., alum). In some embodiments, a metal- hydroxide is iron-hydroxide. A skilled artisan will recognize any number of metal-hydroxide may be successfully utilized in accordance with the present disclosure. Tags [0179] In some embodiments, a fusion polypeptide of the present disclosure comprises an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal-hydroxide binding polypeptide whose amino acid sequence includes a plurality of phosphorylation sites, so that it can adopt phosphorylated and unphosphorylated forms. In some embodiments, a fusion poypeptide of the present disclosure comprises an ending sequence. In some embodiments, an ending sequences comprises a tag. A tag, as used herein, is an amino acid sequence that, when detected and/or measured in a particular sample, indicates that the protein to which it was linked is present in the sample and can provide a quantitative measurement thereof. A variety of tags known in the art may be used in accordance with the present disclosure. For example, in some embodiments, a tag comprises a FLAG tag, a polyhistidine tag, a V5 tag, a MBP tag. [0180] In some embodiments, a tag is inserted at the N- or C-terminus of the fusion polypeptide to minimize interference with fusion polypeptide function. In some embodiments, the tags are be placed internally in-frame within the fusion polypeptide sequence without affecting functionality. Preferred locations for the tag may be determined in part based on available empirical data (e.g., from affinity tag or other fusion polypeptide experiments such as polyhistidine, FLAG tag, MBP tag, etc.), three dimensional structures of the fusion polypeptide or similar polypeptides and/or in vivo or in vitro expression experiments. [0181] In some embodiments, a tag can also contain a short sequence motif, such as an affinity tag or chromatography tag to allow for partial or complete purification from a complex mixture or preparation. In some embodiments, the peptide tags can be designed to allow inclusion of a fixed number (e.g., one, two, three, or more) of a set of preselected (e.g., one, two, three, or more) amino acids. Production of fusion polypeptide metal-hydroxide complexes [0182] In one aspect of the present disclosure, fusion polypeptide-metal hydroxide complexes described herein are produced by (1) making a fusion polypeptide; (2) phosphorylating a fusion polypeptide; and (3) contacting a fusion polypeptide with metal hydroxide. Production of fusion polypeptides [0183] In some embodiments, fusion polypeptides described herein are made in host cells using techniques for exogenous expression. Methods of exogenously expressing polypeptides are well known in the art and the skilled artisan would recognize a variety of techniques could be successfully utilized. [0184] Recombinant polynucleotides (e.g., DNA or RNA) encoding for fusion polypeptides of the present disclosure may be prepared by a variety of methods available. For example, sequences encoding for fusion polypeptides may be excised from DNA using restriction enzymes, may be amplified from plasmids or genomic polynucleotide sequences using, for example, polymerase chain reaction, or may be synthesized using chemical synthesis techniques. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide encoding for fusion polypeptides of the present disclosure. [0185] Recombinant polynucleotides encoding fusion polypeptides of the present disclosure may be cloned into a vector capable of expressing a fusion polypeptide. Cloning may be carried out according to a variety of methods available (e.g., Gibson assembly, restriction digest and ligation, etc.). In some embodiments, a vector is a viral vector. In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid. [0186] In some embodiments, a vector capable of expression comprises a recombinant polynucleotide that encodes a fusion polypeptide of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of recombinant polynucleotides that encode a plurality polypeptides. In some embodiments, a plurality of recombinant polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic.). In some embodiments, a plurality of recombinant polypeptides are expressed, each of which is expressed from a separate vector. [0187] In some embodiments, a vector capable of expression comprising a recombinant polynucleotide encoding a fusion polypeptide of the present disclosure is used to express a fusion polypeptide in a host cell. A host cell may be selected from a variety of the available and known host cells (e.g., Human Embryonic Kidney (HEK) cells, suspension HEK293 cells, Chinese Hamster Ovary cells) suitable expressing fusion polypeptides disclosed herein. [0188] A variety of methods are available to introduce a vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction. [0189] In some embodiments, a transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art. Phosphorylating fusion polypeptides [0190] In some embodiments, fusion polypeptides described herein are phosphorylated by contacting a fusion polypeptide with a kinase. In some embodiments, a fusion polypeptide is contacted with a kinase by co-expressing a fusion polypeptide in a host cell with a kinase. In some embodiments, co-expression is achieved by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell. In some embodiments, co-expression is achieved by introducing a single, multi-cistronic (e.g., bi- cistronic) vector that comprises a plurality of recombinant polynucleotides. In some embodiments, a recombinant polynucleotides encode a fusion polypeptide and a recombinant polynucleotide encoding a kinase. In some embodiments, a transformed host cell is cultured following introduction of a vector into a host cell. Without wishing to be bound by any one theory, upon co-expression of a fusion polypeptide and a kinase in a host cell, a kinase can contact a fusion polypeptide, phosphorylating it. [0191] In some embodiments, co-expression is achieved by introducing two vectors, one comprising a recombinant polynucleotide encoding a fusion polypeptide and one comprising a recombinant polynucleotide encoding a kinase, into a host cell. In some embodiments, two vectors are introduced at a ratio of vector encoding fusion polypeptide to vector encoding a kinase introduced into a host cell optimized to achieve a desired, relative level of expression of fusion polypeptide to kinase. In some embodiments, a ratio of vector encoding fusion polypeptide to vector encoding a kinase is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30: 1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1. [0192] In some embodiments, co-expression is achieved by introducing one vector comprising both a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polypeptide encoding a kinase (e.g., a bi-cistronic vector) into a host cell. In some embodiments, a recombinant polynucleotide encoding a fusion polypeptide and a recombinant polynucleotide encoding a kinase are operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, multiple sequences that control expression (e.g., promoters) are utilized to achieve a desired ratio of expression of fusion polypeptide to kinase. In some embodiments, a ratio is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30: 1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.. [0193] Exemplary nucleotide and amino acid sequences of Fam20C kinases include those described in Table 3. Table 3: Exemplary nucleotide and amino acid sequences of Fam20C kinases Complex formation [0194] In some embodiments, a fusion polypeptide described herein, when exposed to a metal-hydroxide (e.g., aluminum hydroxide) forms a complex therewith. In some embodiments, fusion polypeptides comprise hydroxyl replacement groups (e.g., phosphate groups) for adsorption via ligand exchange with a metal hydroxide. In some embodiments, a fusion polypeptide can, via electrostatic interactions, form a complex with a metal hydroxide. [0195] In some embodiments, a fusion polypeptide metal-hydroxide complex of the present disclosure is formed by mixing. In some embodiments, mixing occurs in a buffer (e.g., a tris-buffered saline buffer). In some such embodiments, a buffer does not contain phosphate. In some such embodiments, a buffer does not contain a substance or substances that solubilize a metal-hydroxide (e.g., citric acid, malic acid, or lactic acid). Without wishing to be bound by any one theory, buffers that contain phosphate compete with, and can hinder, complex formation. In some embodiments, mixing occurs for a duration of time at a particular temperature. In some such embodiments, a duration of time is 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, or 45 minutes. In some such embodiments, a particular temperature is approximately 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C. Purification [0196] Commonly aberrant products (e.g., residual protein, host cell contaminants, etc.) are removed from fusion polypeptide preparations. A variety of purification technologies are available and known to the skilled artisan. [0197] In some embodiments, a fusion polypeptide can be purified by a chromatography method. In some embodiments, such a chromatography purification method can be performed with a chromatography method known in the art, including, but not limited to, high-performance liquid chromatography, size exclusion chromatography, ion exchange chromatography, wherein components of a mixture travel through a stationary phase at different speeds, resulting in separation from one another. It will be apparent to a skilled artisan a variety of solid substrates may be used (e.g., beads, particles, microspheres, resins, etc.). For example, in some embodiments, a solid substrate has properties such that, in accordance with the present disclosure, permits a different retention time for a fusion polypeptide relative to any other undesirable components in the preparation of fusion polypeptide. [0198] In some embodiments, a fusion polypeptide can be purified by an affinity- based purification method. In some embodiments, such an affinity-based purification method may be performed with a solid substrate known in the art. A wide variety of substrates could be utilized, such as, for example, membranes, polymeric beads, magnetic beads, or composites. For example, in some embodiments, a solid substrate (e.g., polymeric beads or particles) coated or pre-charged with a substance or composition (e.g., nickel ions) that has a high binding affinity for a fusion polypeptides can be useful in accordance with the present disclosure such that a fusion polypeptide will bind to a solid substrate, while any other undesirable components present in a preparation will remain in solution. In some embodiments, a fusion polypeptide may be eluted from a solid substrate. In some embodiments, elution may be carried out using specific elution. For example, in some embodiments specific elution is completed by challenging a polypeptide-substrate complex by an agent or agents that will compete for complexation with either a substrate or a polypeptide, releasing a polypeptide into solution. In some embodiments, elution may be carried out using non-specific elution. For example, in some embodiments, non-specific elution is completed by manipulating solvent or buffer conditions (e.g., increasing concentration of a buffer, e.g., an imidazole buffer) to reduce the associate rate constant, resulting in dissociation of the polypeptide from the substrate. Characterization [0199] Among other things, in some embodiments, the present disclosure provides technologies for characterizing fusion polypeptides (e.g., phosphorylated or unphosphorylated preparations thereof) and/or of complexes comprising such fusion polypeptides and metal hydroxides. In characterization is performed during and/or following production process. In some embodiments, a particular preparation process may be modified or terminated in light of a characterization (e.g., if a particular preparation fails to meet one or more specifications). In some embodiments, such characterization may involve assessment of one or more of metal-hydroxide retention, degree of phosphorylation, heterogeneity of phosphorylation, signaling activity, and/or efficacy. Exemplary characterization of phosphate content [0200] In some embodiments, degree of phosphorylation (e.g., of fusion polypeptides of the present disclosure and/or preparations thereof) is characterized. A variety of methods are available for measurement of degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide). For example, in some embodiments, degree of phosphorylation can be determined by a colorimetric method. In some embodiments, a colorimetric method is or comprises a malachite green assay. Without wishing to be bound by any one theory, a malachite green assay is based on quantification of a green complex formed between Malachite green, molybdate, and free orthophosphate which can be measured (e.g., using a spectrophotometer or plate reader). [0201] In some embodiments, degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 0.5-10, 0.5-9, 0.5-8, 1-10, 1-9, 1-8, 2-10, 2-9, 2-8, 3-10, 3-9, 3- 8, 4-10, 4-9, 4-8, 5-10, 5-9, 5-8, 6-10, 6-9, or 6-8. In some embodiments, degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) is 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7.7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10. [0202] In some embodiments, heterogeneity of phosphorylation of fusion polypeptides of the present disclosure and/or preparations thereof are characterized. In some embodiments, heterogeneity of phosphorylation is a measurement of the degree of phosphorylation within a given preparation of fusion polypeptide. In some embodiments, heterogeneity of phosphorylation is a measurement of the degree of phosphorylation across a plurality of preparations of fusion polypeptide. In some embodiments, heterogeneity of phosphorylation is a measurement of location of particular phosphate groups on a polypeptide within a given preparation of fusion polypeptide. In some embodiments, heterogeneity of phosphorylation is a measurement of location of particular phosphate groups on a polypeptide across a plurality of preparations of fusion polypeptide. [0203] A variety of technologies is available for measurement of heterogeneity of phosphorylation. For example, in some embodiments, degree of phosphorylation can be determined by a chromatography method. In some embodiments, a chromatography method comprises ion-exchange chromatography. In some embodiments, for example, a chromatography method comprises analytical anion-exchange chromatography. Anion- exchange chromatography is a form of ion exchange where a negatively charged biomolecule (e.g., a phosphorylated form of a fusion polypeptide disclosed herein) binds to a positively charged resin. In some embodiments, anion-exchange chromatography can be used to resolve polypeptides with different numbers of phosphorylated amino acid residues (e.g., differentially phosphorylated polypeptides). Without wishing to be bound by any one theory, polypeptide phosphorylation confers variability in a polypeptide’s charge, permitting separation of differentially phosphorylated polypeptides using ion-exchange chromatography (e.g., anion-exchange chromatography). Use of a gradient elution buffer (e.g., a buffer with increasing salt concentrations) to elute from the ion exchange (e.g., anion exchange) column permits separation of differentially phosphorylated polypeptides. In some embodiments, a buffer is, for example, a Tris buffer. In some embodiments, a linear gradient of Tris buffer is utilized. In some embodiments, a linear gradient of Tris buffer comprises over a linear gradient from 20 mM Tris, pH 7.1 to 20 mM Tris, 525 mM NaCl, pH 7.1 over a pre-defined period of time. In some embodiments, a linear gradient is conducted over a period of 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, 40 minutes, or longer. [0204] In some embodiments, differentially phosphorylated polypeptides are dephosphorylated. In some embodiments, dephosphorylation comprises use of a phosphatase (e.g., a lambda phosphatase). In some embodiments, a fusion polypeptide is incubated with a phosphatase for a period of time and at a temperature that permits activity of said phosphatase and dephosphorylation of said fusion polypeptide. In some embodiments, dephosphorylation occurs at an incubation temperature of approximately 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or higher. In some embodiments, dephosphorylation occurs for an incubation time of 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, or longer. In some embodiments, dephosphorylation occurs for an incubation time of 25-65 minutes, 30-60 minutes, 35-55 minutes, 40-50 minutes, 30-65 minutes, 35-65 minutes, 40-65 minutes, 45- 65 minutes, 50-65 minutes, or 55-65 minutes. [0205] In some embodiments, differentially phosphorylated polypeptides are dephosphorylated prior to separation. In some embodiments, differentially phosphorylated polypeptides of the present disclosure are assessed relative to an appropriate reference standard (e.g., a dephosphorylated and/or non-phosphorylated form of a fusion polypeptide). [0206] In some embodiments, after separation of differentially phosphorylated polypeptides (e.g., by ion-exchange chromatography), the amount of each differentially phosphorylated polypeptide is measured. In some embodiments, the amount of each differentially phosphorylated polypeptide is measured according to a variety of methods available in the art. In some embodiments, for example and without limitation, differentially phosphorylated polypeptides are measured using a malachite green assay, analytical ion exchange, spectrophotometer, colorimetric assays, and/or western blot. Exemplary characterization of metal-hydroxide retention [0207] In some embodiments, fusion polypeptides of the present disclosure, when exposed to a metal-hydroxide (e.g., aluminum hydroxide) forms a complex therewith. In some embodiments, retention of a fusion polypeptide of the present disclosure on a metal- hydroxide (e.g., metal-hydroxide retention) is characterized. A variety of methods are available to measure metal-hydroxide retention. In some embodiments, for example and without limitation, metal-hydroxide retention can be measured by ellipsometry, surface plasmon resonance, optical waveguide lightmode spectroscopy, attenuated total internal reflectance-infrared spectroscopy, circular dichroism spectroscopy (CD), total internal reflectance-infrared spectroscopy (TIRF), and other high resolution microscopy techniques. [0208] In some embodiments, metal-hydroxide retention is characterized using an in vitro assay. For example, fusion polypeptides at a known concentration are mixed with an excess of metal-hydroxide. The concentration of free, non-complexed fusion polypeptides is quantified and compared to a standard curve to determine metal-hydroxide retention. The concentration of free, non-complexed fusion polypeptide can be assessed according to a variety of method known to those of skill in the art. For example, and without limitation, in some embodiments, free, non-complexed, fusion polypeptides are quantified by enzyme- linked immunosorbent assay (ELISA), western blot, bicinchoninic acid assay, or Bradford assay. [0209] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of fusion- polypeptide, when mixed with a metal-hydroxide, forms a complex therewith (e.g., is retained). Exemplary characterization of signaling activity [0210] In some embodiments, fusion polypeptides (and/or complexes thereof) as described herein are characterized for activity (e.g., signaling activity). In some embodiments, activity is characterized by assessing signaling activity (e.g., signaling competency) compared to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and/or a fusion polypeptide lacking a metal-hydroxide binding polypeptide. [0211] A variety of methods are available to assess signaling competency. In some embodiments, for example, signaling competency is assessed using an in vitro- or in vivo- based activity assay. [0212] In some embodiments, signaling activity is assessed with an in vitro activity assay. In some embodiments, an in vitro activity assay comprises measuring activation or inhibition of downstream signaling of a fusion polypeptide. In some embodiments, measuring activation or inhibition of downstream activity comprises use of a reporter (e.g., a reporter assay). In some embodiments, a reporter assay measures activity using a detectable molecule (e.g., a reporter) that correlates with fusion polypeptide activity. [0213] In some embodiments, a reporter comprises a fluorescent, bioluminescent, and/or other detectable probe known to those of skill in the art. In some embodiments, a reporter comprises use of a gene reporter. A gene reporter, for example, can be activated upon signaling elicited from a polypeptide. For example, upon activation of gene reporter transcription, a detectable product or enzyme that can be activated upon addition of substrate, generating a detectable product and/or by-product, can be utilized. In some embodiments, an enzyme useful in accordance with a reporter assay is, for example, luciferase or an alkaline phosphatase (e.g., secreted alkaline phosphatase, SEAP). In some such embodiments, a HEK-blue-IL12 reporter assay is utilized. [0214] In some embodiments, signaling activity is assessed with an in vivo activity assay. In some embodiments, a fusion polypeptide is administered to a subject (e.g., a mouse, non-human primate, human, etc.) and activity is assessed. In some embodiments, activity is assessed, for example, by measuring activation or inhibition of downstream signaling of a fusion polypeptide as compared to an appropriate reference standard (e.g., activity of a wild-type polypeptide). A variety of methods are available to measure activation or inhibition of downstream signaling of a fusion polypeptide. For example, and without limitation, differential gene expression, protein expression, and/or alterations in post-translational modifications induced by a fusion polypeptide can be measured. Exemplary efficacy characterization [0215] In some embodiments, efficacy can be characterized according to a variety of methods that are available. In some embodiments, for example, a fusion polypeptide (or complex thereof) as described herein is administered (e.g., by intratumoral or peritumoral injection) to a subject (e.g., mouse, non-human primate, human, etc.) and efficacy is determined in comparison to an appropriate reference standard. An appropriate reference standard can be, for example, a wild-type polypeptide and/or a polypeptide lacking a metal- hydroxide binding polypeptide, or having a metal-hydroxide binding polypeptide in a non- binding (e.g., non-phosphorylated) state. [0216] In some embodiments, efficacy is determined pre-clinically in an animal model (e.g., in mice, rats, non-human primates, etc.). In some embodiments, a fusion polypeptide is administered (e.g., by intratumoral or peritumoral injection) to an animal model. For example, in some embodiments, an animal model is an animal model with a tumor (e.g., an animal model of cancer). In some embodiments, a cancer animal model is generated by inoculating said animal model with tumor cells. In some embodiments, an animal model is inoculated with tumor cells at the flank region. In some embodiments, an animal model is inoculated with tumor cells in a clinically relevant region (e.g., a mammary fat pad). [0217] In some embodiments, an animal model of cancer is administered a fusion polypeptide of the present disclosure. In some embodiments, an animal model of cancer is administered a reference standard (e.g., a wild-type polypeptide and/or a polypeptide lacking a metal-hydroxide binding polypeptide). In some embodiment, a variety of available, pre- determined measurements for efficacy known in the art, such as, for example, tumor volume and/or percent survival are assessed over time relative to an appropriate reference standard (e.g., a wild-type polypeptide and/or a polypeptide lacking a metal-hydroxide binding polypeptide). [0218] In some embodiments, efficacy of a fusion polypeptide is determined clinically. In some embodiments, a fusion polypeptide is administered (e.g., by intratumoral, peritumoral injection, or into a tumor-draining lymph node) to a subject with a tumor. In some embodiments, a variety of available, pre-determined measurements for efficacy known in the art, such as, for example, tumor volume and/or percent survival are assessed over time relative to a subject with a tumor administered reference standard (e.g., a treatment in the art of known efficacy and/or placebo). Compositions Preparations of fusion polypeptides [0219] In some embodiments, the present disclosure, among other things, provides fusion polypeptide preparations. Fusion polypeptide preparations are preparations comprising a fusion polypeptide that, in some embodiments, is purified from a cell culture production of said fusion polypeptide described herein. In some embodiments, a fusion polypeptide preparation comprises an unphosphorylated form of a fusion polypeptide. In some embodiments, a fusion polypeptide preparation comprises a phosphorylated form of the fusion polypeptide. In some embodiments, a fusion polypeptide preparation comprises a mixture of both unphosphorylated and phosphorylated forms of a fusion polypeptide. [0220] In some embodiments, a fusion polypeptide preparation is a preparation comprising pharmaceutical-grade fusion polypeptide. In some embodiments, a fusion polypeptide preparation is a preparation comprising fusion polypeptide which its one or more characterization attributes are assessed and determined to meet a release and/or acceptance criteria (e.g., as described herein). Examples of such product quality attributes include, but are not limited to, degree of phosphorylation and/or heterogeneity of phosphorylation. [0221] In some embodiments, a fusion polypeptide preparation comprises a phosphorylated form of a fusion polypeptide and a kinase used to phosphorylate a fusion polypeptide (e.g., as described herein). In some embodiments, a kinase is removed from a fusion polypeptide preparation. [0222] In some embodiments, a phosphorylated fusion polypeptide preparation comprises fusion polypeptides with varying degrees of phosphorylation. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of a preparation of fusion polypeptide is 0.5-7, 1-7, 2-7, 3-7, 4-7, 5-7, 6-7, 0.5-6, 0.5-5, 0.5-4, 1-6, 2-6, 3-6, 4-6, 5-6, 0.5-10, 0.5-9, 0.5-8, 1-10, 1-9, 1-8, 2-10, 2-9, 2-8, 3-10, 3-9, 3-8, 4-10, 4-9, 4-8, 5-10, 5-9, 5-8, 6-10, 6-9, or 6-8. In some embodiments, the degree of phosphorylation (e.g., the average number of phosphate molecules per polypeptide) of a preparation of fusion polypeptide is 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 7.10, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9 or 10. [0223] In some embodiments, the present disclosure, among other things, provides a fusion polypeptide preparation comprising a fusion polypeptide-metal hydroxide complex. In some embodiments, a preparation comprising a fusion polypeptide-metal hydroxide complex comprises any of a variety of suitable metal-hydroxide known in the art. For example, and without limitation, a metal-hydroxide may be an aluminum hydroxide or an iron hydroxide. In some embodiments, a fusion polypeptide-metal hydroxide complex comprises a mass ratio of fusion polypeptide to metal hydroxide (e.g., aluminum hydroxide), for example as defined by metal (e.g., aluminum) mass. In some embodiments, the ratio, is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. [0224] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition comprising a fusion polypeptide disclosed herein. In some embodiments, the pharmaceutical composition is formulated as a fusion polypeptide-metal hydroxide complex. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and/or adjuvant. [0225] In some embodiments, acceptable pharmaceutical composition formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, formulation material(s) are for subcutaneous and/or intravenous administration. In some embodiments, formulation material(s) are for local administration (e.g., intratumoral or peritumoral administration). In some embodiments, a pharmaceutical composition can contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of fusion polypeptides. In some embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants. (Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed., Mack Publishing Company (1995). In some embodiments, a pharmaceutical composition comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mM NAOAC, pH 5.2, 9% Sucrose. In some embodiments, an optimal pharmaceutical composition will be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In some embodiments, such compositions may influence the physical state, stability, rate of in vivo release and rate of in vivo clearance of a fusion polypeptide-metal hydroxide complex. [0226] In some embodiments, a primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, in some embodiments, a suitable vehicle or carrier can be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. In some embodiments, the saline comprises isotonic phosphate-buffered saline. In some embodiments, neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In some embodiments, pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which can further include sorbitol or a suitable substitute therefore. In some embodiments, a composition comprising a fusion polypeptide metal hydroxide complex or a fusion polypeptide is prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, in some embodiments, a composition comprising a fusion polypeptide-metal hydroxide complex or a fusion polypeptide is formulated as a lyophilizate using appropriate excipients such as sucrose. Use Methods of treatment [0227] In one aspect, the present disclosure relates to methods of treating a subject with a medical condition. In some embodiments, the present disclosure relates to methods of treating a subject with a tumor (e.g., a subject with cancer). In general, methods of treatment are aimed at reducing tumor volume, reducing and/or preventing metastases, prolonging survival, and/or curing the condition. Appropriate subjects or individuals receiving a fusion polypeptide or fusion polypeptide metal hydroxide complex of the present disclosure include, for example, humans or other mammals (e.g., mice, rats, rabbits, dogs, horses, cats, pigs, or non-human primates) that have a tumor (e.g., cancer). [0228] In some embodiments, a method of treating a subject with a tumor comprises a step of: treating a subject with a complex comprising: a fusion polypeptide comprising an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal- hydroxide binding polypeptide and a metal hydroxide. In some embodiments, a method of treating a subject with a tumor comprises administering a fusion polypeptide comprising: an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal- hydroxide binding polypeptide, wherein a fusion polypeptide is formulated with a metal hydroxide. [0229] In some embodiments, a complex as described herein is administered as a monotherapy. In some embodiments, a complex as described herein is administered in combination with a second therapeutic. In some embodiments, a complex as described herein is administered to a subject wherein a subject has received or is receiving therapy with at least one additional therapeutic. [0230] Fusion polypeptides and/or preparations and/or complexes thereof of the present disclosure, among other things, are useful for treating a subject with a tumor. Non- limiting examples of diseases associated with a tumor include cancer (e.g., carcinoma, sarcoma, metastatic diseases or hematopoietic neoplastic disorders). A tumor, including a metastatic tumor, can arise from a plurality of primary tumor types. For example, and without limitation, in some embodiments, a tumor or metastatic tumor can arise from a primary tumor of the kidney (e.g., renal cell carcinoma), head and neck (e.g., head and neck squamous cell carcinoma), prostate, breast (e.g., triple-negative), colon, skin (e.g., melanoma, merkel cell carcinoma, cutaneous T-cell lymphoma, cutaneous squamous cell carcinoma, basal cell carcinoma), lung (e.g., non-small cell lung cancer), and pancreas. Accordingly, fusion polypeptides and preparations thereof disclosed herein, including fusion polypeptide metal-hydroxide complexes and preparations thereof, can be administered to subject who has cancer. [0231] It will be appreciated by those skilled in the art that amounts of a fusion polypeptide-metal hydroxide complex, fusion polypeptide or a preparation thereof sufficient to reduce tumor growth and size, or a therapeutically effective amount, will vary not only on the particular compounds or preparations selected, but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient, and will ultimately be at the discretion of the patient's physician or pharmacist and/or based upon clinical guidelines. The length of time during which the compounds used in the instant method will be given varies on an individual basis and/or be based upon clinical guidelines. [0232] In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises a step of treating the subject with a complex comprising a fusion polypeptide comprising an immunomodulatory polypeptide that comprises an immune agonist moiety and a metal-hydroxide binding polypeptide and a metal hydroxide. In some embodiments, a fusion polypeptide and a metal-hydroxide are formulated together. Formulated together, for example, comprises a pre-formed complex of fusion polypeptide and metal-hyrdoxide. In some embodiments, a fusion polypeptide and metal-hydroxide are mixed immediately prior to administration. [0233] In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered by intratumoral injection. In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered by peritumoral injection. In some embodiments, a method of treating a subject with a tumor (e.g., cancer) comprises treating a subject with a complex wherein a complex is administered to a tumor-draining lymph node or lymph nodes. [0234] Methods of the present invention often involve administration of a therapeutically effective amount of a particular agent. A therapeutically effective amount is an amount sufficient to achieve (in principle, for a subject of comparable characteristics, such as species, body type, size, extent of disease or disorder, degree or type of symptoms, history of responsiveness, and/or overall health) an intended biological or medical response or therapeutic benefit in a tissue, system or subject. For example, a desirable response may include one or more of: delaying or preventing the onset of a medical condition, disease or disorder, slowing down or stopping the progression, aggravation, or deterioration of the symptoms of the condition, bringing about ameliorations of the symptoms of the condition, and curing the condition. [0235] When combinations of therapeutic agents are administered, the amount of any individual agent required in the combination may be different from the amount required of that same agent to achieve its therapeutic effect alone. In some cases, synergies between or among therapeutic agents used in a combination may reduce amounts required; in other cases, inhibitory interactions may increase amounts required. Thus, in general, therapeutically effective amounts of a combination of agents may utilize different absolute amounts of the agents than constitute therapeutically effective amounts of the agents individually. Combination therapies [0236] In some embodiments, fusion polypeptide metal-hydroxide complexes or preparations thereof as disclosed herein are administered in combination with a second therapeutic agent. A second therapeutic agent may be selected from a variety of available anti-tumor agents known in the art. In some embodiments, a second therapeutic agent is administered prior to administration of a fusion polypeptide metal-hydroxide complex. In some embodiments, a second therapeutic agent is administered concurrently with a fusion polypeptide metal-hydroxide complex. In some embodiments, a second therapeutic agent is administered after administration with a fusion polypeptide metal-hydroxide complex. [0237] For example, in some embodiments, a second therapeutic is radiation (e.g., ionizing radiation). In some embodiments, an amount of ionizing radiation administered is between about 1 Gy and about 1 ,000 Gy, about 5 Gy and about 900 Gy, about 10 Gy to about 800 Gy, about 10 Gy to about 700 Gy, about 10 Gy to about 600 Gy, about 10 Gy to about 500 Gy, about 10 Gy to about 400 Gy, about 10 Gy to about 300 Gy, about 10 Gy to about 200 Gy, about 10 Gy to about 100 Gy, about 5 Gy and about 15 Gy, between about 7.5 Gy and about 12 Gy, or between about 10 Gy and about 12 Gy. In some embodiments, an amount of ionizing radiation administered is about 12 Gy. In some embodiments, an amount of ionizing radiation is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1 ,000 Gy. In some embodiments, an amount of ionizing radiation is less than about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy. [0238] For example, in some embodiments, a second therapeutic agent is a chemotherapeutic agent. In some embodiments, a chemotherapeutic agent may be a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In some embodiments, a chemotherapeutic agent may be any approved chemotherapeutic agent. For example, and without limitation, a chemotherapeutic agent can be one or more of adriamycin, anastrozole, cyclophosphamide, docetaxel, doxifluridine, doxorubicin, erlotinib, fluorouracil, gemcitabine, imatinib, iressa, letrozole, methotrexate, paclitaxel, tarceva, and trastuzumab. A chemotherapeutic agent may be administered according to any approved and/or known regimen in the art. [0239] For example, in some embodiments, a second therapeutic agent is an anti- tumor antibody. In some embodiments, an anti-tumor antibody is an immune modulator. In some embodiments, an immune modulator is a checkpoint inhibitor. In some embodiments, a checkpoint inhibitor is an antibody or a functional fragment thereof. In some embodiments, an antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and/or LAG3. In some embodiments, an antibody targets PD-1 (e.g., pembrolizumab). An anti-tumor antibody may be administered according to any approved and/or known regimen in the art. [0240] For example, in some embodiments, a second therapeutic agent is a surgical tumor resection. In some embodiments, a fusion polypeptide metal-hydroxide complex is administered prior to surgical tumor resection. In some embodiments, a fusion polypeptide metal-hydroxide complex is administered to tissue after tumor resection, which tissue may include, for example, remaining tumor (e.g., tumor cells). In some embodiments, a fusion polypeptide metal-hydroxide complex is administered to tissue which cannot be removed by surgical tumor resection, or tissue proximal to the resection, during said resection. [0241] For example, in some embodiments, a second therapeutic agent is or comprises cell therapy. In some embodiments, a cell therapy is or comprises natural killer (NK) cells. In some embodiments, a cell therapy is or comprises tumor infiltrating lymphocytes (TILs). In some embodiments, a cell therapy is or comprises macrophages or other myeloid cells. In some embodiments, a cell therapy is or comprises cells that have been expanded ex vivo. In some embodiments, a cell therapy is or comprises Chimeric Antigen Receptor (CAR) effector cell therapy (e.g., CAR T cells). CARs are genetically- engineered, artificial transmembrane receptors, which confer a selected specificity for a ligand of choice onto an immune effector cell (e.g. a T cell, natural killer cell or other immune cell) and which results in activation of the effector cell upon recognition and binding to the ligand. Often, such ligand specificity is achieved by engineering the antigen specificity of a monoclonal antibody into the CAR, thereby targeting the CAR T cell to the antigen recognized by the antibody. [0242] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g,. CAR-T cells) are cells that are derived (e.g., isolated) from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with an arbitrary specificity to a ligand. The cells perform at least one effector function (e.g. induction of cytokines) that is stimulated or induced by the specific binding of the ligand to the CAR and that is useful for treatment of the same patient's disease or condition. The effector cells may be T cells (e.g. cytotoxic T cells or helper T cells). One skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, cells other than T cells ( e.g,. natural killer cells, stem cells, etc) may be engineered to express CARs, so that a chimeric antigen receptor effector cell may comprise an effector cell other than a T cell. In some embodiments, a CAR effector cell is a T cell (e.g. a cytotoxic T cell); in some embodiments, such CAR-T cell exerts its effector function (e.g. a cytotoxic T cell response) on a target cell when brought in contact or in proximity to the target or target cell (e.g. a cancer cell) (see e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).In some embodiments, a cell therapy (e.g., a CAR effector cell therapy) utilizes of Tumor Infiltrating Lymphocytes (TILs). TILs target cancer cells. In some embodiments, TILs are isolated from a subject with cancer and expanded ex vivo. In some such embodiments, TILs are isolated and expanded ex vivo after surgical resection of the tumor. In some embodiments, before administration of TILs, a subject is treated with a lymphodepleting conditioning regimen (Rohaan, Maartje W et al. “Adoptive cellular therapies: the current landscape.” Virchows Archiv : an international journal of pathology vol.474,4 (2019): 449-461). [0243] In some embodiments, a cell therapy (e.g., a CAR effector cell therapy) utilizes Natural Killer (NK) cells. Natural killer (NK) cells are an essential part of tumor immunosurveillance, evidenced by higher cancer susceptibility and metastasis in association with diminished NK activity in mouse models and clinical studies. In some embodiments, for example, using an array of germline-encoded surface receptors, NK cells are able to recognize and rapidly act against malignant cells without prior sensitization (iu, S., Galat, V., Galat4, Y. et al. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 14, 7 (2021)). [0244] In some embodiments, fusion polypeptide metal-hydroxide complexes or preparations thereof as disclosed herein are administered to a subject who has received or is receiving a therapy with at least one additional therapeutic. An additional therapeutic agent may be selected from a variety of anti-tumor agents known in the art. In some embodiments, an additional therapeutic agent is administered prior to administration of a fusion polypeptide metal-hydroxide complex. In some embodiments, an additional therapeutic agent is administered concurrently with a fusion polypeptide metal-hydroxide complex. In some embodiments, an additional therapeutic agent is administered after administration with a fusion polypeptide metal-hydroxide complex. [0245] For example, in some embodiments, an additional therapeutic is radiation (e.g., ionizing radiation). In some embodiments, an amount of ionizing radiation administered is between about 1 Gy and about 1 ,000 Gy, about 5 Gy and about 900 Gy, about 10 Gy to about 800 Gy, about 10 Gy to about 700 Gy, about 10 Gy to about 600 Gy, about 10 Gy to about 500 Gy, about 10 Gy to about 400 Gy, about 10 Gy to about 300 Gy, about 10 Gy to about 200 Gy, about 10 Gy to about 100 Gy, about 5 Gy and about 15 Gy, between about 7.5 Gy and about 12 Gy, or between about 10 Gy and about 12 Gy. In some embodiments, an amount of ionizing radiation administered is about 12 Gy. In some embodiments, an amount of ionizing radiation is greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1 ,000 Gy. In some embodiments, an amount of ionizing radiation is less than about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, or 50 Gy. [0246] For example, in some embodiments, an additional therapeutic agent is a chemotherapeutic agent. In some embodiments, an additional therapeutic agent is or comprises a targeted therapy (e.g., BRAF inhibitor, MEK inhibitor, etc.). In some embodiments, a chemotherapeutic agent may be any approved chemotherapeutic agent. For example, and without limitation, a chemotherapeutic agent can be one or more of adriamycin, anastrozole, cyclophosphamide, docetaxel, doxifluridine, doxorubicin, erlotinib, fluorouracil, gemcitabine, imatinib, iressa, letrozole, methotrexate, paclitaxel, tarceva, and trastuzumab. A chemotherapeutic agent may be administered according to any approved and/or known regimen in the art.In some embodiments, an additional therapeutic agent is an anti-tumor antibody. In some embodiments, an anti-tumor antibody is an immune modulator. In some embodiments, an immune modulator is a checkpoint inhibitor. In some embodiments, a checkpoint inhibitor is an antibody or a functional fragment thereof. In some embodiments, an antibody targets one or more of PD-1, PD-L1, CTLA-4, TIM3, TIGIT, and/or LAG3. In some embodiments, an antibody targets PD-1 (e.g., pembrolizumab). An anti-tumor antibody may be administered according to any approved and/or known regimen in the art. For example, in some embodiments an additional therapeutic agent is or comprises cell therapy. In some embodiments, a cell therapy is or comprises Chimeric Antigen Receptor (CAR) effector cell therapy (e.g., CAR T cells). CARs are genetically-engineered, artificial transmembrane receptors, which confer a selected specificity for a ligand of choice onto an immune effector cell (e.g. a T cell, natural killer cell or other immune cell) and which results in activation of the effector cell upon recognition and binding to the ligand. Often, such ligand specificity is achieved by engineering the antigen specificity of a monoclonal antibody into the CAR, thereby targeting the CAR T cell to the antigen recognized by the antibody. [0247] In some embodiments, chimeric antigen receptor-expressing effector cells (e.g,. CAR-T cells) are cells that are derived (e.g., isolated) from a patient with a disease or condition and genetically modified in vitro to express at least one CAR with an arbitrary specificity to a ligand. The cells perform at least one effector function (e.g. induction of cytokines) that is stimulated or induced by the specific binding of the ligand to the CAR and that is useful for treatment of the same patient's disease or condition. The effector cells may be T cells (e.g. cytotoxic T cells or helper T cells). One skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, cells other than T cells ( (e.g,. natural killer cells, stem cells, etc) may be engineered to express CARs, so that a chimeric antigen receptor effector cell may comprise an effector cell other than a T cell. In some embodiments, a CAR effector cell is a T cell (e.g. a cytotoxic T cell); in some embodiments, such CAR-T cell exerts its effector function (e.g. a cytotoxic T cell response) on a target cell when brought in contact or in proximity to the target or target cell (e.g. a cancer cell) (see e.g., Chang and Chen (2017) Trends Mol Med 23(5):430-450).In some embodiments, a cell therapy (e.g., a CAR effector cell therapy) utilizes of Tumor Infiltrating Lymphocytes (TILs). TILs target cancer cells. In some embodiments, TILs are isolated from a subject with cancer and expanded ex vivo. In some such embodiments, TILs are isolated and expanded ex vivo after surgical resection of the tumor. In some embodiments, before administration of TILs, a subject is treated with a lymphodepleting conditioning regimen (Rohaan, Maartje W et al. “Adoptive cellular therapies: the current landscape.” Virchows Archiv : an international journal of pathology vol.474,4 (2019): 449-461). [0248] In some embodiments, a cell therapy (e.g., a CAR effector cell therapy) utilizes Natural Killer (NK) cells. Natural killer (NK) cells are an essential part of tumor immunosurveillance, evidenced by higher cancer susceptibility and metastasis in association with diminished NK activity in mouse models and clinical studies. In some embodiments, for example, using an array of germline-encoded surface receptors, NK cells are able to recognize and rapidly act against malignant cells without prior sensitization (iu, S., Galat, V., Galat4, Y. et al. NK cell-based cancer immunotherapy: from basic biology to clinical development. J Hematol Oncol 14, 7 (2021)). [0249] In some embodiments, a cell therapy (e.g., a CAR effector cell therapy) comprises myeloid cells. In some embodiments, myeloid cells are or comprise macrophages. Macrophages have been shown to take up alum.
Exemplification Example 1: Optimization of exemplary metal-hydroxide binding polypeptides (e.g., alum binding polypeptide (ABP)) sequence for increased phosphorylation and aluminum hydroxide binding Methods [0250] Gene synthesis - Genes were synthesized at ATUM Bio (Newark, CA) and cloned into the pD2610-v10 vector. Human Fam20C (Uniprot Q8IXL6) was cloned with its native signal peptide and a linker-KDEL sequence (GGGSKDEL) for intracellular retention fused at the c-terminus. Single-chain human IL12 constructs were cloned with a human IL2 signal peptide and mature human IL12B/p40 (Uniprot P29460) fused to mature human IL12A/p35 (Uniprot P29459) through a (G4S)3 linker. Single-chain mouse IL12 constructs were cloned with a human IL2 signal peptide and mature mouse IL12B/p40 (Uniprot P43432) fused to mouse IL12A/p35 (Uniprot P43431) through a (G4S)3 linker. Various alum binding polypeptide (ABP) sequences were genetically fused to the c-terminus of mouse and human IL12 followed by a His6 tag for affinity purification. [0251] Polypeptide expression and purification - Plasmids encoding human or mouse IL12 constructs were transiently transfected in suspension HEK-293 cells either with or without co-transfection with the human Fam20C-KDEL plasmid. In most co- transfections, a 4:1 mass ratio of IL12 plasmid to Fam20C-KDEL plasmid was used. Supernatants were harvested and IL12 fusion polypeptides purified by affinity chromatography on NiSepharose Excel (Cytiva 17-3712-02). Eluted fusion polypeptides were formulated in Tris-buffered saline (TBS), pH 7.4 and purity determined on a Perkin Elmer GXII capillary electrophoresis system. Polypeptide aggregation was assessed by HPLC-SEC with a 300^ pore size. Where needed, proteins were further purified by FPLC- SEC on a HiLoad 16/600 or 26/600 Superdex 200 pg column (Cytiva 28-9893-36) and monomeric peak fractions were pooled. [0252] In some cases, fusion polypeptides were further polished by anion exchange chromatography to enrich for highly phosphorylated species. Fusion polypeptides in TBS were diluted 3-fold in WFI and loaded on a HiTrap Q Sepharose column. Samples were washed with 1% Tx-13 in 0.33x TBS followed by 15 column volumes of 20 mM Tris, pH 8.1. Samples were eluted with a linear gradient from 20 mM Tris, pH 8.1 to 20 mM Tris, pH 8.1, 600 mM NaCl over 20 column volumes. Selected fractions were pooled and buffer exchanged into TBS. [0253] Malachite green assay - The average number of phosphate molecules per polypeptide was determined using the Pierce Phosphoprotein Estimation Assay Kit (23270) according to manufacturer’s instructions. The phosphorylated protein standard Phosvitin was resuspended in TBS and diluted from 100 to 2.5 ug/mL to generate a standard curve. Each test polypeptide was diluted in TBS to 300 μg/mL and 100 μg/mL. 50 μL of each standard or test agent was mixed with 50 μL of 2.0N NaoH in a flat bottom, 96 well plate (Griener 655101) for alkaline hydrolysis of phosphate from seryl and threonyl residues. Samples were incubated at either 65 o C for 30 minutes or 37 o C for 1 hour, then neutralized by adding 50 μL of 4.7N HCL to each well and mixing for 30 seconds on a shaker. 50 μL of phosphate reagent comprised of one volume ammonium molybdate solution and 3 volumes of malachite green solution was then added to each well and mixed for 30 seconds. Samples were incubated at room temperature for 30 minutes, then absorbance measured at 650 nm. The number of phosphate molecules per test agent was derived based on the known phosphate content in the Phosvitin standard curve. [0254] Aluminum hydroxide (alum) retention assay - Binding and retention of fusion polypeptides to aluminum hydroxide was tested in vitro. Test polypeptides at a final concentration of 100-250 μg/mL in TBS were mixed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass (Invivogen Cat# alu-vac-250) or TBS only as a control to a final volume of 50 μL. Fusion polypeptide/alum mixtures were resuspended thoroughly by pipetting and incubated at room temperature for 30 minutes. Halfway through the incubation, the mixtures were resuspended again by pipetting. The fusion polypeptide/alum mixtures or polypeptide only controls were then diluted 20x in elution buffer containing a final concentration of 1 mM phosphate, 20% mouse serum to a final volume of 1 mL. Diluted samples were incubated at 37 o C with gentle rotating for 24-48 hours. At each timepoint, 50 μL of sample was removed and centrifuged at 18,000xg for 10 minutes to pellet the aluminum hydroxide. Cleared supernatant was transferred to a new tube and stored at 4 o C until ready for analysis. The concentration of free polypeptide in each supernatant sample was quantified using a mouse IL12p70 ELISA kit (R&D Systems m1270). All dilutions were made in TBS + 1% BSA + 0.1% Tween-20. Test agents were used for standard curves with a top concentration of 1 ng/mL and 2x dilutions and supernatant samples were diluted to a theoretical concentration of 1 ng/mL and 0.5 ng/mL if all polypeptide was released. [0255] SAX-10 HPLC assay - Fusion polypeptide phosphorylation was also assessed by analytical anion-exchange chromatography on a Thermo ProPac SAX-10 column (4 x 250 mM, 10 μm). Samples were diluted 4-fold in 20 mM Tris, pH 7.1 and loaded on the column at a 1.0 mL/min flow rate. Samples were eluted over a linear gradient from 20 mM Tris, pH 7.1 to 20 mM Tris, 525 mM NaCl, pH 7.1 over 28 minutes. In some cases, fusion polypeptides were dephosphorylated with lambda protein phosphatase (New England Biolabs, P0753L) prior to running on the column. 50 μg of fusion polypeptide was incubated with 10x PMP buffer, 10 mM MnCL2, and 2-4 μL phosphatase at 30 o C for 30-60 minutes. [0256] Syngeneic tumor models - B16F10 cells were grown in DMEM + 10% FBS at 37 o C, 5% CO2. C57BL/6 mice aged 7-9 weeks were inoculated with 1x10 6 B16F10 cells in 0.1 mL of PBS solution at the right flank region. When tumors reached ~75 mm 3 , mice were randomized and injected intratumorally with 20 μL vehicle or 5-7.7 μg mIL12-ABP that had been mixed and pre-incubated with 25-50 μg aluminum hydroxide as defined by metal mass for 30 minutes at room temperature. In some groups, 200 μg anti-PD1 (Bioxcell BP0273) in a 50 μL volume was injected intraperitoneally on days 0, 3, 6, and 9. Tumor volume and body weight were measured 3x weekly for the duration of the study and mice were euthanized when their tumor volume reach 2000 mm 3 . In some studies, two tumors were inoculated in each animal with 1x10 6 B16F10 cells injected in the right flank and 1x10 5 cells injected in the left flank. In these dual flank studies, the mIL12-ABP/alum complex or controls were injected in the larger right flank tumor only. Results [0257] Single-chain human and mouse IL12 constructs were generated containing the mature IL12B and IL12A sequences linked by a (G4S)3 linker with a c-terminal His6 tag for affinity purification. Alum binding polypeptide containing variants (IL12-ABP) were cloned by inserting nucleotide sequences encoding various ABP sequences c-terminal to IL12 and before the His tag. A first set of human and mouse IL12-ABP fusion polypeptides were transiently expressed in 100 mL HEK cultures either with or without co-transfection with a plasmid encoding human Fam20C kinase with a c-terminal KDEL sequence for intracellular retention. Fusion polypeptides were purified by Ni-NTA chromatography and average phosphorylation levels per fusion polypeptide assessed by malachite green assay (Figure 3A). hIL12-ABP and mIL12-ABP fusion polypeptides co-expressed with Fam20C (denoted as “-K”) had significantly higher phosphate levels compared to fusion polypeptides without Fam20C co-expression or wild-type hIL12 or mIL12 lacking the ABP co-expressed with Fam20C. While all ABP sequences were phosphorylated, fusion polypeptides with the ABP10 sequence had reproducibly higher phosphorylation levels than the other ABP variants tested. [0258] Mouse IL12-ABP variants were expressed at 1 L scale in transient HEK with Fam20C co-expression and purified by sequential Ni-NTA and SEC chromatography. In the malachite green assay, mIL12-ABP10 again had the highest phosphorylation followed in order by ABP20-G4-GS20, ABP20-G4, and ABP20 (Figure 3B). Mouse IL12-ABP fusion polypeptides were then tested in a dual flank B16F10 syngeneic tumor model in combination with systemic PD-1 blockade. 5 μg mIL12 or mIL12-ABP was mixed with 25 μg aluminum hydroxide as defined by metal mass and incubated for 30 minutes prior to injection into the right flank tumor on Day 6 post tumor implantation. All mIL12-ABP / alum complexes delayed tumor growth compared to PD-1 alone or mIL12 + PD-1, but the greatest delays were observed with mIL12-ABP10 suggesting that increased phosphorylation can improve in vivo efficacy after a single injection when complexed with alum (Figures 4A-4G). [0259] To further enhance ABP phosphorylation, alum retention, and in vivo efficacy, a larger panel of mIL12-ABP constructs was generated with ABP sequences listed in Figure 5 differing in number of SXE sites, spacing of SXE sites, flanking residues, and linker sequences. Polypeptides were co-transfected with Fam20C in HEK cells and purified by Ni-NTA chromatography. Phosphorylation levels of the purified polypeptides were characterized in the malachite green assay (Figure 6A and Figure 6B). While all ABP’s had significantly higher phosphorylation compared to negative controls, the highest phosphorylation levels were detected in mIL12 fusion polypeptides comprising ABP10, ABP20-G8-GS, ABP20-G4-6x, and ABP20-G4-8x. [0260] To reduce the chance of off-target phosphorylation of additional serines in the ABP sequence, the linkers for ABP20-G4-6x and ABP20-G4-8x were changed from GGGGSGGGG to GGGGEGGGG and the serine immediately upstream of the His-tag was removed. These new sequences were referred to as ABP20-G4-6x-GE and ABP20-G4-8x- GE (Figure 5). mIL12 fused to ABP10, ABP20-G4-6x-GE, or ABP20-G4-8x-GE was co- expressed with Fam20C at 1 L scale in transient HEK. Fusion polypeptides were purified by sequential Ni-NTA affinity and size exclusion chromatography. All fusion polypeptides ran as a single peak on SDS-PAGE and SEC demonstrating that there was no significant degradation or aggregation (Figures 7A-7C). The three exemplary fusion polypeptides were tested in an alum retention assay in which fusion polypeptides were incubated with a 10-fold mass excess of aluminum hydroxide as defined by metal mass for 30 minutes in TBS then diluted in elution buffer containing 5 mM phosphate and 20% mouse serum for 24 hours. While all 3 fusion polypeptides had significantly greater retention on alum compared to unmodified IL12, mIL12-ABP20-G4-6x-GE was eluted more quickly than both mIL12- ABP10 and mIL12-ABP20-G4-8x-GE (Figure 8). [0261] Following transient co-transfection with Fam20C in HEK cells, there is potential for heterogeneity in phosphate levels between IL12-ABP molecules within a given fusion polypeptide sample. In order to generate more homogeneously phosphorylated material, mIL12-ABP10 and mIL12-ABP20-G4-8x-GE were further purified by anion exchange chromatography which is able to separate different phospho-species on the basis of the added negative charge with more heavily phosphorylated fusion polypeptides binding tighter to the column and eluting later. Samples were eluted with a linear salt gradient and the second half of each elution peak was collected and pooled to eliminate early eluting fractions containing lower levels of phosphorylation (Figures 9A-9B). The enriched pools of mIL12-ABP10 and mIL12-ABP20-G4-8x-GE after preparative anion exchange chromatography were compared to the original material by analytical ion exchange chromatography (Figures 10A-10C). An additional sample of each fusion polypeptide was enzymatically dephosphorylated with phosphatase and run as a control. Prior to anion exchange polishing, both samples have an early eluting fraction that partially overlaps with the dephosphorylated trace. However, after ion exchange polishing, the samples have a more homogeneous profile that elutes later than the dephosphorylated fusion polypeptide suggesting enrichment for fusion polypeptides with higher phosphorylation. [0262] Ion-exchange enriched mIL12-ABP10 and mIL12-ABP20-G4-8x-GE were tested in the alum retention assay along with a sample of mIL12-ABP20-G4-8x-GE prior to ion-exchange polishing (Figure 11A). Polypeptides were complexed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass then diluted in elution buffer containing 5 mM phosphate and 20% mouse serum for 24 hours before quantifying free polypeptide in the supernatant. The ion exchange purified mIL12-ABP20-G4-8x-GE had the highest retention with 89% bound to alum at 24 hours compared to 86% for mIL12- ABP10 and 78% for the non-ion exchanged material demonstrating that the low phosphate fractions elute off the alum faster. In a second alum retention assay, samples were bound to a 10-fold mass excess of aluminum hydroxide as defined by metal mass then eluted with 5 mM phosphate and 20% mouse serum for up to 48 hours (Figure 11B). The ion exchange purified mIL12-ABP20-G4-8x-GE again had the highest retention with 82% bound to alum at 48 hours compared to 78% for mIL12-ABP10 and 17% for unmodified mIL12. Example 2: Cellular activity of exemplary fusion-polypeptide-metal hydroxide complex Methods [0263] HEK-Blue-IL12 potency assay - In vitro IL12 signaling activity was assessed using the HEK-Blue-IL12 reporter assay (Invivogen hkb-il12) according to manufacturer’s instructions. This cell line is derived from HEK293 cells stably transfected with human IL12Rβ1 and hIL12Rβ2 and a secreted alkaline phosphatase (SEAP) reporter under the control of a STAT4 inducible promoter. Since mouse IL12 cross-reacts with the human IL12 receptors, this cell line can be used to assess potency of both human and mouse IL12 derived constructs. HEK-Blue-IL12 cells were cultured in DMEM + 4.5 g/l glucose, 2 mM L-glutamine, 10% heat inactivated FBS, Pen-Strep (100 U/mL) and 100 ug/mL Normocin and passaged at 70-80% confluency. For potency testing, the same media was used without Normocin. [0264] Test agents or IL12 controls were diluted in assay media to generate a titration series with a top concentration of 10 μg/mL and 3x dilutions. For samples mixed with alum, fusion polypeptides at a final concentration of 50 μg/mL were mixed with a 10x mass excess of aluminum hydroxide as defined by metal mass in TBS and incubated at room temperature for 30 minutes with shaking before diluting in assay media as above. 20 μL of each sample in the titration series was transferred to a 96 well plate and mixed with 180 μL of HEK-Blue-IL12 cell suspension (280,000 cells/mL) for a final top fusion polypeptide concentration of 1 μg/mL and 50,000 cells/well. Plates were then incubated overnight at 37 o C in 5% CO2. The next day, 20 μL of supernatant from each well was transferred to a new plate and mixed with 180 μL of QUANTI-Blue solution (Invivogen rep-qbs), a colorimetric reagent than turns blue in the presence of secreted alkaline phosphatase. Plates were incubated for at 37 o C for 3 hours, then absorbance measured at 620-655 nm. [0265] In some experiments, test agents in TBS were mixed with aluminum hydroxide to a final concentration of 200 μg/mL fusion polypeptide and 2 mg/mL aluminum hydroxide as defined by metal mass, then incubated at RT for 30 minutes. Mixtures were then diluted 5x in elution buffer to a final concentration of 40 μg/mL fusion polypeptide with 1 mM phosphate and 20% mouse serum and incubated at 37 o C with rotating for 24 hours. Samples were centrifuged at 18,000xg at 4 o C for 10 minutes to pellet alum and the supernatant carefully removed and saved. Pellets were resuspended in an equal volume of elution buffer. Supernatant and resuspended alum pellets were then diluted in assay media and tested for activity in the HEK-Blue-IL12 assay as described above. Results [0266] mIL12-ABP10 and mIL12-ABP20-G4-8x-GE were tested in a HEK-Blue- IL12 reporter assay that expresses SEAP in response to IL12 induced signaling. The IL12- ABP fusion polypeptides were titrated either alone or after mixing with a 10-fold mass excess of aluminum hydroxide as defined by metal mass in TBS and incubating for 30 minutes. Both fusion polypeptides were active in the assay with EC50 values of 3.6 and 5.4 ng/mL for mIL12-ABP10 and mIL12-ABP20-G4-8x-GE, respectively, compared to 3.5-5.4 ng/mL for unmodified mIL12 (Figures 12A-12B). When bound to alum the EC50 values shifted ~2-4 fold with EC50 values of 8 ng/mL for mIL12-ABP10 and 22 ng/mL for mIL12- ABP20-G4-8x. The potency shift is likely a product of steric hindrance in the way the IL12 is presented while complexed with aluminum hydroxide. [0267] To assess activity of the alum bound and eluted fractions, mIL12-ABP20-G4- 8x-GE was complexed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass then incubated in elution buffer containing 1 mM phosphate and 20% mouse serum for 24 hours. The alum was then pelleted by centrifugation, supernatant removed, and the pellet resuspended in an equal volume of elution buffer. The supernatant fraction, resuspended alum pellet, and a control sample of mIL12-ABP20-G4-8x-GE incubated overnight in elution buffer without alum were tested in the HEK-Blue-IL12 assay. While the control sample without alum had a similar EC50 to previous measurements at 7 ng/mL, the supernatant fraction had an EC50 >1000 ng/mL demonstrating that minimal IL12 eluted off the alum during the extended incubation. In contrast, the alum pellet was active in the assay with a potency shift of ~5x demonstrating that IL12 remains active while retained on alum for extended time (Figure 13). Example 3: Use of fusion-polypeptide-metal hydroxide complex as monotherapy and in combination therapy Methods [0268] Syngeneic tumor models - CT26 cells were cultured in RPMI1640 + 10% FBS. BALB/c mice aged 7-9 weeks were inoculated subcutaneously with 5x10 5 CT26 cells in 0.1 mL PBS at the right flank region. When tumors reached ~75 mm 3 , mice were randomized and injected intratumorally with 20 μL vehicle or 5 μg mIL12-ABP that had been mixed and pre-incubated with 50 μg aluminum hydroxide as defined by metal mass for 30 minutes at room temperature. Tumor volume and body weight were measured 3x weekly for the duration of the study and mice were euthanized when their tumor volume reach 2000 mm 3 . Tumor volumes were measured in two dimensions using a caliper, and the volume was calculated using the formula: V = (L x W x W)/2, where V is tumor volume, L is tumor length (the longest tumor dimension) and W is tumor width (the longest tumor dimension perpendicular to L). [0269] B16F10 cells were grown in DMEM + 10% FBS at 37 o C, 5% CO 2 . C57BL/6 mice aged 7-9 weeks were inoculated with 1x10 6 B16F10 cells in 0.1 mL of PBS solution at the right flank region. When tumors reached ~75 mm 3 , mice were randomized and injected intratumorally with 20 μL vehicle or 7.7 μg mIL12-ABP that had been mixed and pre- incubated with 50 μg aluminum hydroxide as defined by metal mass for 30 minutes at room temperature. In some groups, 200 μg anti-PD1 (Bioxcell BP0273) in a 50 μL volume was injected intraperitoneally on days 0, 3, 6, and 9. Tumor volume and body weight were measured 3x weekly for the duration of the study and mice were euthanized when their tumor volume reach 2000 mm 3 . [0270] 4T1 cells were grown in RPMI1640 + 10% FBS at 37 o C, 5% CO 2 . BALB/c mice aged 7-9 weeks were inoculated in the right mammary fat pad with 3x10 5 4T1 cells in 0.1 mL PBS. When tumors reached ~75 mm 3 , mice were randomized and injected intratumorally with 20 μL vehicle or 5 μg mIL12-ABP that had been mixed and pre- incubated with 50 μg aluminum hydroxide as defined by metal mass for 30 minutes at room temperature. Tumor volume and body weight were measured 3x weekly. On day 28 post- tumor inoculation, mice were euthanized and metastases counted in the lung. Results [0271] mIL12-ABP10 and mIL12-ABP20-G4-8x-GE were compared in multiple syngeneic tumor models. Fusion polypeptides were mixed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass in TBS and incubated at RT for 30 minutes to form a fusion polypeptide-aluminum hydroxide complex prior to injecting in the animals. In the CT26 colorectal cancer model, a single intratumoral injection of 5 μg of either mIL12- ABP or mIL12-ABP20-G4-8x-GE fusion polypeptides complexed with 50 μg alum as defined by metal mass on Day 6 post tumor inoculation led to significant tumor delays and regressions compared to vehicle treated mice. Complete responses with no measurable tumor were observed in 4/10 mice treated with mIL12-ABP10 + alum and 5/10 mice treated with mIL12-ABP20-G4-8x-GE + alum (Figures 14A-14C). [0272] In a second study in the refractory B16F10 tumor model, 7.7 μg of either mIL12-ABP10 or mIL12-ABP20-G4-8x-GE complexed with 50 μg alum as defined by metal mass was injected intratumorally on Day 6 and 13 following tumor inoculation. In some animals, anti-PD1 antibody was also administered intraperitoneally on Day 0, 3, 6, & 9. Both mIL12-ABP agents complexed with alum significantly delayed tumor growth compared to vehicle. The tumor delay was further extended in groups receiving the combination treatment with systemic PD-1 blockade. In both the monotherapy and PD-1 combination groups, the median survival was longer in mice treated with mIL12-ABP20- G4-8x-GE compared to mIL12-ABP10 (Figures 15A-15E). [0273] mIL12-ABP20-G4-8x-GE was also tested in an orthortopic 4T1 model that forms spontaneous lung metastases (Figures 16A-16B). Tumor-bearing mice were treated with a single intratumoral injection of 5 μg of mIL12-ABP fusion polypeptide complexed with 50 μg alum as defined by metal mass on Day 7 post tumor inoculation. mIL12-ABP20- G4-8x-GE /alum treatment led to growth delay of the primary tumor compared to IT injection of vehicle (Figure 16A). On Day 28, animals were sacrificed and metastases counted in the lung. While 10/10 vehicle treated mice had at least one lung metastasis, 6/10 of the mice in the mIL12-ABP20-G4-8x-GE /alum treated mice were metastases-free demonstrating that local injection of the mIL12-ABP/alum complex had also induced anti- tumor effects against distal, non-injected lesions (Figure 16B). Example 4: Identification of an optimal level of phosphorylation on mIL12-ABP [0274] mIL12-ABP20-G4-8x-GE was co-expressed with Fam20C-KDEL transiently in HEK as described above. Fusion polypeptide was purified by Ni-NTA chromatography and size exclusion chromatography, then run on a HiTrap Q Sepharose anion exchange column with a linear salt elution gradient. Individual elution fractions were collected and phosphate levels measured by malachite green assay. Average phosphate levels per fraction ranged from 2 to 7 with lower phosphorylated fusion polypeptide eluting earlier and highly phosphorylated fractions retained longer on the column (Figures 17A-17B). Individual fractions were then assessed in the alum retention assay. Fusion polypeptides were bound to a 10-fold mass excess of aluminum hydroxide as defined by metal mass then eluted in solution containing 1 mM phosphate and 50% mouse serum. There was a clear trend where fractions eluting earlier off the anion exchange resin and containing lower phosphorylation levels had lower retention on alum compared to later eluting fractions with higher phosphorylation levels. Fraction 110 with 2.4 phosphates per fusion polypeptide had <50% fusion polypeptide retained on alum at 24 hours and fraction 111 with 3.7 phosphates per fusion polypeptide had ~80% fusion polypeptide retained on alum. Fractions 112 and 113 with 4.6 and 5.8 phosphates per fusion polypeptide respectively were highly retained with >95% bound to alum at 24 hours. Fractions 114-117 with >6 phosphates per fusion polypeptide had no measurable free fusion polypeptide eluting off alum in the assay (Figures 18A-18B). [0275] Individual fractions were also tested in the HEK-Blue-IL12 reporter assay either alone or complexed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass (Figures 19A-19D). Fractions 110 and 112 had similar peak activity levels with or without alum and 2-3 fold shifts in EC50 when bound to alum. In contrast, fraction 114 and 117 had reduced peak activity when complexed with alum. This suggests that beyond a certain point, excess phosphorylation on the fusion polypeptide can decrease functional activity in the presence of alum [0276] Together, these data, summarized in Figure 20, suggest that there may be an optimal levels of phosphorylation per murine fusion polypeptide at ~4-6 with less than that having insufficient alum retention and >6 losing activity in functional assays. Example 5: Production and characterization of tagless human IL12-ABP20-G4-8x-GE [0277] Genes were synthesized encoding single-chain human IL12-ABP20-G4-8x- GE comprising mature human IL12B/p40 (Uniprot P29460) fused to mature human IL12A/p35 (Uniprot P29459) through a (G 4 S) 3 linker with the ABP20-G4-8x-GE peptide at the c-terminus. Unlike the mouse constructs, the fusion polypeptides were cloned without the c-terminal His tag, instead ending in a single serine residue. Human IL12-ABP20-G4- 8x-GE constructs and Fam20C-KDEL genes were cloned into a single vector with two expression cassettes with different promoters selected to express the hIL12-ABP20-G4-8x- GE fusion polypeptide at an 8-fold higher level than Fam20C-KDEL. Fusion polypeptides were stably transfected in CHO cells using the ATUM Leap-In-Transposase system and stable pools selected. After 14 day expression in a fed-batch culture, supernatants were harvested and purified by multiple chromatography steps including anion exchange chromatography capture and size exclusion chromatography polishing. After purification, the untagged hIL12-ABP20-G4-8x-GE was >97% pure as measured by SDS-PAGE and SEC. [0278] Human IL12-ABP20-G4-8x-GE is active in the HEK-Blue-IL12 assay with an EC50 of 3.6 ng/mL alone or 8.2 ng/mL when complexed with aluminum hydroxide (Figure 21A). Following incubation of the fusion polypeptide /alum complex in elution buffer containing 1 mM phosphate and 20% mouse serum, the supernatant EC50 shifts >300-fold suggesting minimal fusion polypeptide elution after 24 hours, while the pellet maintained activity with a 5x EC50 shift (Figure 21B). In a separate alum retention assay, hIL12 or hIL12-ABP was bound to a 10-fold mass excess of aluminum hydroxide as defined by metal mass then diluted in elution buffer containing 1 mM phosphate and 20% mouse serum with free fusion polypeptide detected by ELISA (Figure 21C). ~80% of the hIL12- ABP20-G4-8x-GE is retained on the alum at 24 hours compared to 0% for unmodified IL12. [0279] A second lot of human IL12-ABP20-G4-8x-GE was prepared as described above and further purified by anion exchange chromatography and eluted with a linear salt gradient. Individual elution fractions were collected and phosphate levels were measured by malachite green assay. Average phosphate levels per fraction ranged from 5.8 to 8.8 with lower phosphorylated fusion polypeptide eluting earlier and highly phosphorylated fractions retained longer on the column (Figure 22). [0280] Individual fractions were assessed in an alum retention assay in which fusion polypeptides were bound to a 10-fold mass excess of aluminum hydroxide as defined by metal mass then eluted in solution containing 1 mM phosphate and 40% serum (Figure 23). Similar to the murine IL12-ABP20-G4-8x-GE protein, fractions of human IL12-ABP20-G4- 8x-GE eluting earlier off the anion exchange resin and containing lower phosphorylation levels had lower retention on alum compared to later eluting fractions with higher phosphorylation levels. [0281] Individual fractions were also tested in the HEK-Blue-IL12 reporter assay either alone or complexed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass (Figure 24). In the absence of aluminum hydroxide complexation, all tested fractions had similar potency in the assay with EC50 values between 3.4 – 3.6 ng/mL. Following aluminum hydroxide complexation, earlier eluting fractions with lower phosphorylation had lower EC50 values (5 and 6.4 ng/mL for fraction 645 and 650, respectively), while later eluting fractions with higher phosphorylation levels had more significant shifts in the EC50 (8.6 and 14 ng/mL for fractions 655 and 660, respectively). All tested fractions had similar maximal activity in the assay either with or without aluminum hydroxide complexation. [0282] Together, fractions 650-660 were identified as having a particularly beneficial balance of alum retention and IL12 signaling activity. Thus, preparations characterized by about 5.8 phosphates or more (e.g., about 5.8 to more than about 8.4) per fusion polypeptide showed particularly desirable properties. Additional assessments may be performed to confirm such properties, including, for example, further in vitro and/or in vivo assessments. In some such studies, fractions 650-660 (or other reasonably comparable preparations – e.g., from another batch, or produced via a different process but achieving comparable phosphorylation characteristics, etc.) may be pooled or otherwise combined for assessment; alternatively or additionally, these fractions (or other reasonably corresponding preparations) may be separately assessed. Example 6: Production and characterization of canine IL12-ABP20-G4-8x-GE-His [0283] The present Example documents production and activity of a beneficially phosphorylated preparation of an IL-12 fusion polypeptide as described herein, and of metal hydroxide complexes thereof. Among other things, the present Example documents that preparations of this fusion polypeptide which are characterized by about 5.5 phosphates or more (e.g., about 5.5 to more than about 8.3) per fusion polypeptide showed a particularly desirable balance of alum-binding and IL-12 signaling activities. [0284] An exemplary gene cassette encoding for single-chain canine-IL12-ABP20- G4-8x-GE was synthesized at ATUM Bio and cloned into the pD2610-v5 expression vector. The exemplary canine IL12-ABP20-G4-8x-GE sequence comprises mature canine IL12B/p40 (Uniprot Q28268) fused to mature canine IL12A/p35 (Uniprot F1PPC0) through a (G4S)3 linker with the ABP20-G4-8x-GE peptide and His tag at the C-terminus. The exemplary construct was transiently co-transfected in suspension HEK-293 cells with a human Fam20C-KDEL plasmid at a 4:1 mass ratio. Supernatants were harvested and IL12 fusion polypeptides purified by affinity chromatography on NiSepharose Excel resin (Cytiva 17-3712-02) followed by FPLC-SEC on a HiLoad 16/600 Superdex 200 pg column (Cytiva 28-9893-36) to remove aggregates. The exemplary canine IL12-ABP20-G4-8x-GE protein was further polished by anion exchange chromatography on a HiTrap Q Sepaharose column to enrich for differentially phosphorylated species and eluted with a linear salt gradient. Selected fractions were assessed for numbers of phosphate per protein using a malachite green assay, IL12 signaling potency using a HEK-Blue-IL12 assay, and aluminum hydroxide binding using an alum retention assay with protocols described above (Figure 25). [0285] Average phosphate levels in each fraction ranged from 0.4 to 11.3 PO 4 /protein with lower phosphorylated proteins eluting earlier and highly phosphorylated proteins retained longer on the column. Individual fractions were then assessed in an alum retention assay in which polypeptides were bound to a 10-fold mass excess of aluminum hydroxide as defined by metal mass then eluted in solution containing 1 mM phosphate and 40% serum with free protein quantified over time using a canine IL12p40 ELISA kit (R&D Systems). A trend was observed where fractions eluting earlier off the anion exchange resin and containing lower phosphorylation levels had lower retention on alum compared to later eluting fractions with higher phosphorylation levels (Figure 26). Fractions 91 & 92 with 2.0 and 4.5 PO 4 /protein respectively had the lowest retention with 28% of the IL12 polypeptide in fraction 91 and 65% in fraction 92 retained on the alum at 24 hours. Fractions 93 and 93.5 with 5.5 and 6.5 phosphates per fusion polypeptide respectively were highly retained with >95% bound to alum at 24 hours. Fractions 94-98 with >6.5 phosphates per fusion polypeptide had no measurable free fusion polypeptide eluting off alum in the assay. [0286] Individual fractions were also tested in a HEK-Blue-IL12 reporter assay either alone or complexed with a 10-fold mass excess of aluminum hydroxide as defined by metal mass (Figure 27). Fractions had similar EC50 values when tested as free protein but different potency when complexed on alum with fractions containing high levels of PO4 having larger increases in EC50 after alum complexation. Fractions 93-94.5 were identified as having a particularly beneficial balance of alum retention and IL12 signaling activity. Thus, preparations characterized by about 5.5 phosphates or more (e.g., about 5.5 to more than about 8.3) per fusion polypeptide showed particularly desirable properties. Additional assessments may be performed to confirm such properties, including, for example, further in vitro and/or in vivo assessments (e.g., in an appropriate animal system – e.g., in this case, in dogs). In some such studies, fractions 93-94.5 (or other reasonably comparable preparations – e.g., from another batch, or produced via a different process but achieving comparable phosphorylation characteristics, etc.) may be pooled or otherwise combined for assessment; alternatively or additionally, these fractions (or other reasonably corresponding preparations) may be separately assessed. Example 7: Exemplary canine studies [0287] The present Example documents further assessment of technologies of the present disclosure (e.g., IL-12 fusion polypeptides, fusion polypeptide preparations). For example, the present Example provides further confirmation and/or assessment of phosphate content, metal-hydroxide retention, signaling activity, efficacy, and/or potency, etc.. Among other things, the present Example documents assessment and/or further confirmation of use of IL-12 fusion polypeptides and fusion polypeptide preparations of the present disclosure in a subject, including in a non-human subject, including, for example, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a horse, a sheep, cattle, a primate, and/or a pig. Technologies of the present disclosure (e.g., IL-12 fusion polypeptides, fusion polypeptide preparations) are assessed using any suitable animal model known in the art (see, e.g., Paoloni M et al. Defining the Pharmacodynamic Profile and Therapeutic Index of NHS-IL12 Immunocytokine in Dogs with Malignant Melanoma. PLoS One.2015;10(6):e0129954; Cutrera J et al. Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro-chemo-gene therapy. J Cell Mol Med.2015;19(3):664-675; Cutrera J et al. Safety and efficacy of tumor-targeted interleukin 12 gene therapy in treated and non-treated, metastatic lesions. Curr Gene Ther.2015;15(1):44-54; Von Rueden SK et al. Cancer- Immunity Cycle and Therapeutic Interventions- Opportunities for Including Pet Dogs With Cancer. Front Oncol.2021;11:773420. Published 2021 Nov 19).
Equivalents [0289] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims: