Protein-enclosing polymeric complexes

Abstract

The present disclosure provides, in part, a polymeric complex comprising: (i) an immunocomplex comprising: (a) a cytokine; and (b) a soluble cytokine-binding protein; and (ii) a block copolymer disclosed herein (e.g., a block copolymer represented by Formula (I)).

Description

[0001] DESCRIPTION

[0002] TITLE OF INVENTION

[0003] PROTEIN-ENCLOSING POLYMERIC COMPLEXES

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] The present application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 571,420, filed March 28, 2024, and 63 / 696,708, filed September 19, 2024, the disclosures of each of which are hereby incorporated by reference in their entireties for all purposes.

[0006] SEQUENCE LISTING

[0007] The present application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML copy, created on February 27, 2025, is named RAR-006WO_SL.xml, and is 332,405 bytes in size.

[0008] TECHNICAL FIELD OF THE INVENTION

[0009] The present invention relates to protein (e.g., immunocomplex)-encapsulating polymeric complexes, and methods of use thereof. All disclosures of the references cited herein are incorporated herein by reference in their entirety.

[0010] BACKGROUND ART

[0011] Superagonists are compounds that display higher receptor signaling output than endogenous agonistic molecules (Schrage, R. et al. Br. J. Pharmacol. 173, 3018-3027 (2016)). Reported superagonists encompass small molecules (e.g., Alix, K. et al. ACS Chem. Neurosci. 7, 1565-1574 (2016)), peptides (e.g., Galloway, S. A. E. et al. Front. Immunol. 10, 407741 (2019)), and proteins (e.g., Wrangle, J. M. et al. Lancet Oncol. 19, 694-704 (2018)). These formulations typically feature carefully designed molecular structures which ensure a superior affinity and stable interaction with target receptors (Chow, E. S. H. et al. Bioorg. Med. Chem. Lett. 12, 1985-1988 (2002)), offering opportunities to treat diseases by a powerful manipulation on therapeutic signaling pathways. However, the clinical implementation of superagonists is often accompanied by safety concerns due to hyperactivation of systemic immunity (Stebbings, R. et al. Curr. Opin. Biotechnol. 20, 673- 677 (2009)). Furthermore, protein superagonists are often multicomponent complexes that can separate during circulation, requiring higher doses to achieve a therapeutic effect. Thus, there is a need for alternate strategies to deliver superagonists at lower doses with reduced toxicity, while still achieving a therapeutic effect.

[0012] SUMMARY OF INVENTION

[0013] In one aspect, the present disclosure provides a polymeric complex comprising: (i) an immunocomplex comprising: (a) a cytokine; and (b) a soluble cytokine-binding protein; and (ii) a block copolymer represented by Formula (I): wherein the variables are as defined herein.

[0014] In some embodiments, the block copolymer represented by Formula (I) is a block copolymer represented by Formula (la):

[0015] In another aspect, provided herein is a pharmaceutical composition comprising the polymeric complex as described herein and a pharmaceutically acceptable excipient.

[0016] Also provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering the complex as disclosed herein or the pharmaceutical composition as described herein to the subject.

[0017] In another aspect, a method of delivering an immunocomplex to a cell, the method comprising contacting the cell with the polymeric complex as described herein, wherein a pH level of a region surrounding the cell permits release of cytokine and a cytokine receptor or cytokine-binding fragment thereof is provided.

[0018] Also provided herein is a method of modulating an immune response in a subject receiving an immunotherapy regimen, the method comprising: (i) administering the complex as described herein to the subject;

[0019] (ii) measuring or having measured a biological property of the subject;

[0020] (iii) changing the course of the immunotherapy regimen based on the measurement of the biological property; thereby modulating the immune response in the subject.

[0021] In another aspect, provided herein is a method of manufacturing the polymeric complex as described herein, the method comprising:

[0022] (a) contacting the cytokine and the soluble cytokine-binding protein, thereby forming the immunocomplex;

[0023] (b) contacting (i) a hydrophilic polymer comprising a reactive group and (ii) a plurality of cyclic monomers comprising a basic group under reaction conditions sufficient for a ring-opening polymerization reaction, thereby forming a product of the ring-opening polymerization reaction;

[0024] (c) contacting the product of the ring-opening polymerization reaction with a charge regulator, thereby forming the block copolymer; and

[0025] (d) contacting the block copolymer with the immunocomplex, thereby forming the polymeric complex.

[0026] BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1A is a schematic illustration of IL- 15 nanosuperagonist (Nano-SA) structure and the assembly principle. The carboxydimethyl-maleic anhydride (CDM) groups modified on the side chain of the polymer can simultaneously bind with IL- 15 and IL-15Rα via pH- sensitive amide bonds formed with amines. Further crosslinks between polymers finally lead to the formation of Nano-SA.

[0028] FIG. IB shows SDS-PAGE of free proteins and Nano-SA. Nano-SA showed significantly reduced mobility in electric field, indicating a giant molecular size.

[0029] FIGs. 1C and ID show fluorescence correlation spectroscopy (FCS) measurements of IL-15cx or Nano-SA. In these samples, IL-15 was labelled with AlexaFluor 647 (A647) and IL-15Rα was labelled with AlexaFluor 488 (A488). Diffusion kinetics of the samples were measured in aqueous buffer with 633 nm and 488 nm excitation lasers separately. Autocorrelation function (ACF) curves of the samples were plotted in FIG. 1C. Diffusion time calculated from ACF was plotted in FIG. ID. Data shown as the mean ± standard deviation (S.D.), n = 3 independent measurements.

[0030] FIG. IE shows a representative transmission electronic microscopy (TEM) image of Nano-SA. The cores of Nano-SA were clearly visualized as black dots.

[0031] FIG. IF shows size distribution of Nano-SA cores observed by TEM. n = 100 particles analysed.

[0032] FIG. 1G shows representative dynamic light scattering (DLS) measurements, showing the distribution of Nano-SA hydrodynamic size in aqueous buffer.

[0033] FIG. 1H shows Z-averaged hydrodynamic diameters of IL-15cx and Nano-SA. Data are shown as the mean ± S.D., n = 3 independent measurements.

[0034] FIG. II show -potentials of IL-15cx and Nano-SA, indicating their surface charge conditions. Data shown as the mean ± S.D., n = 3 independent measurements.

[0035] FIG. 2A is a schematic illustration of a Forster resonance energy transfer (FRET)- based probe system for tracking IL- 15 ex stability. IL- 15 was labelled with A647 (acceptor) and IL-15Rα was labelled with A555 (donor). The FRET signal between IL- 15 and IL-I5Rα could indicate the association state between the two components.

[0036] FIG. 2B shows the time-dependent FRET signal change of IL-15cx or Nano-SA incubated in murine plasma. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0037] FIG. 2C shows representative SDS-PAGE of Nano-SA after incubation in buffer with different pH values.

[0038] FIG. 2D shows hydrodynamic sizes of Nano-SA in buffer at indicated pH. The size was calculated from the diffusion coefficient measured from FCS. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0039] FIG. 2E shows percentages of remaining micelles in buffer at indicated pH. The size was calculated from the diffusion coefficient measured from FCS. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0040] FIG. 2F shows the payload release profile of Nano-SA under different pH conditions. The experiment was performed by dialysis method with membranes of different molecular weight cut-off (MWCO). Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0041] FIG. 2G shows an enzyme-linked immunosorbent assay (ELISA) binding test of IL- 15cx, Nano-SA, and activated Nano-SA samples. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0042] FIG. 2H shows representative fluorescence images of KHYG-1 cells incubated with IL-15cx, Nano-SA, and activated Nano-SA samples. IL- 15 was labelled with A647 and IL- 15Rα was labelled with A488. Nuclei were stained with Hoechst. Scale bar = 100 pm.

[0043] FIG. 21 shows flow cytometry measurement quantifying the fluorescence intensity from the cells in FIG. 2G.

[0044] FIG. 2J shows an in vitro bioactivity evaluation of Nano-SA. KHYG-1 cells were incubated with samples and the cell proliferation was quantified by cell counting kit (CCK)-8 to indicate the response. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0045] FIG. 3A shows blood circulation profiles observed from intravital confocal microscope (IVCLSM). IL-15 was labelled with A647 and IL-15Rα was labelled with A488. Samples were injected to the tail vein of mice. Earlobe skin of the mice was observed by IVCLSM. Scale bar = 50 pm.

[0046] FIG. 3B shows plot profiles of the fluorescence signals in the images presented in FIG. 3A. Signals were quantified along the white arrow on the images.

[0047] FIG. 3C shows time-dependent profiles of fluorescence signals in the vessels (white boxes on the images in FIG. 3A).

[0048] FIG. 3D shows an in vivo evaluation of the stability of IL-15cx by IVCLSM. Mice were injected with the FRET-based probe samples. The earlobe skin was observed by IVCLSM to detect the FRET signal. Scale bar = 50 pm.

[0049] FIG. 3E shows representative ex vivo fluorescence images of the tumors and quantitative evaluation of IL-15 (A647-labelled) and IL-15Rα (A488-labelled) levels in tumors 8 h post-injection. Data are plotted as the mean ± S.D., n = 3 independent samples.

[0050] FIG. 3F shows representative ex vivo fluorescence images of the tissues excised 8 h post injection. FIG. 3G shows quantified biodistribution profile of IL-15 and IL-15Rα in organs and tumors. Data are plotted as the mean ± S.D., n = 3 independent samples. Results were compared via one-way ANOVA.

[0051] FIG. 3H shows relative ratios of IL- 15 to IL-15Rα in organs and tumors. Data are plotted as the mean ± S.D., n = 3 independent samples. Results were compared via one-way ANOVA.

[0052] FIG. 4A is a schematic illustration showing the design of the activation indicator (AL Nano-SA). The polymer was labelled with QSY-21, which is a specific quencher against A647. IL-15 was labelled with A647 and IL-15Rα was labelled with A488. After forming the AI-Nano-SA, the fluorescence emission from A647 was quenched, while the emission from A488 remained as the control. Upon activation, the formulation dissociated and the quenching between QSY-21 and A647 should be relieved, inducing an increased fluorescence from A647.

[0053] FIG. 4B shows time-dependent in vivo fluorescence images of mice bearing tumors after intravenous (i.v.) injection of AI-Nano-SA. Red fluorescence signals from A467-labeled IL- 15 could be observed from the tumor area.

[0054] FIG. 4C shows representative ex vivo fluorescence images of the tissue samples excised 12 h post injection.

[0055] FIG. 4D shows quantification of the Red (A467-labelled IL-15) / Green (A488-labeled IL-15Rα) (R / G) fluorescence intensity ratio from the images depicted in FIG. 4C. Data are plotted as the mean ± S.D., n = 3 groups of samples from 3 independent animals.

[0056] FIG. 4E shows IVCLSM images observing the red (A467-labelled IL- 15) and green (A488-labeled IL-15Rα) fluorescence from earlobe skin (left panel) and tumor tissue (right panel).

[0057] FIG. 4F shows three-dimensional reconstitution of IVCLSM images 6 h postinjection.

[0058] FIG. 5 A is a schematic illustration of an experimental schedule. Mice bearing MC38 tumors received three daily treatments of IL- 15 ex or Nano-SA (intravenous injection (i.v.), 10 μg IL-15cx equivalence (e.q.)). Mice were sacrificed one day after the final treatment for the following analysis. FIG. 5B shows concentrations of cytokines in mice plasma measured by ELISA. Data are plotted as the mean ± S.D., n = 6 independent samples.

[0059] FIG. 5C shows frequency of NK cells out of living cells in blood samples measured by flow cytometry. Data are plotted as the mean ± S.D., n = 4 independent samples.

[0060] FIG. 5D shows representative flow cytometry plots of NK cells stained with a CD69 antibody. Frequency of CD69+ NK cells out of overall NK cells was plotted in the right histogram. Data are plotted as the mean ± S.D., n = 4 independent samples.

[0061] FIG. 5E shows flow cytometry analysis of spleen samples. Frequencies of NK cells out of living cells (left panel) and CD69+ NK cells out of overall NK cells (right panel) were plotted. Data are plotted as the mean ± S.D., n = 4 independent samples.

[0062] FIG. 5F shows representative photographs and weights of spleens excised from mice. Data are plotted as the mean ± S.D., n = 3 independent samples.

[0063] FIG. 5G shows frequencies of NK cells out of living cells in liver samples. Data are plotted as the mean ± S.D., n = 4 independent samples.

[0064] FIG. 5H shows representative fluorescence images of tumor sections stained with NKp46 and CD8α antibodies. The nuclei were stained with Hoechst. Scale bar = 100 pm.

[0065] FIG. 51 shows representative flow cytometry plots of lymphocytes in tumors. The cells were stained with NK1.1 and CD3 antibodies.

[0066] FIG. 5J shows abundances of NK cells, CD8+ T cells , and NKT cells in tumors. Data plotted as the mean ± S.D., n = 4 independent samples. All results were compared via one-way ANOVA between every two groups. The statistically significant difference (p < 0.05) was marked with the p value.

[0067] FIG. 6A shows a schematic illustration of an experimental schedule of Nano-SA monotherapy against subcutaneous (s.c.) MC38 tumors. Mice bearing s.c. MC38 tumors received three treatments of IL-15cx or Nano-SA (i.v., 10 μg IL-15cx e.q.).

[0068] FIG. 6B shows representative images of tumor sections stained by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay. Tumors were harvested on Day 10. Scale bar = 1 mm. FIG. 6C shows independent tumor growth curves in each group, n = 5 independent animals.

[0069] FIG. 6D shows survival curves of mice in different groups, n = 5 independent animals. Mice with tumor volume exceeding 1000 mm3were euthanized and counted as dead.

[0070] FIG. 6E shows efficacy test of Nano-SAin orthotopic (o.t.) MC38 tumors. MC38 cells were inoculated at the cecal wall. Mice were kept until natural death for recording the survival curves, n = 5 independent animals. Results were compared via Logrank test.

[0071] FIG. 6F shows representative fluorescence images of tumor sections after different treatments. The sections were stained with PD-L1 antibodies (yellow) and Hoechst (blue). Scale bar = 500 pm.

[0072] FIG. 6G shows a schematic illustration of the combinational therapy experiments. IL- 15cx or Nano-SA were dosed via i.v. injection (3 μg IL-15cx e.q.). PD-L1 antibody (aPD-L1) was dosed via intraperitoneal (i.p.) injection (100 μg).

[0073] FIG. 6H shows independent tumor growth curves in each group, n = 6 independent animals.

[0074] FIG. 6I shows survival curves of mice in different groups, n = 6 independent animals. Mice with tumor volume exceeding 1000 mm3were euthanized and counted as dead.

[0075] FIG. 6J shows representative fluorescence images of tumor sections after Nano-SA monotherapy or combinational therapy of Nano-SA + aPD-L1. The sections were stained with CD8α antibody and granzyme B (GB). Nuclei were stained with Hoechst. Scale bar = 100 pm.

[0076] FIG. 6K shows tumor growth curves of rechallenge experiment. The cured mice in J were re-inoculated with MC38 cells (s.c., 105cells per mouse).

[0077] FIG. 7 is a1H-NMR of PEG-pLL(CDM) polymer. The length of pLL block was determined by comparing the integration of the peaks of -CH2-CH2on PEG (δ = 3.5 ppm) and -CH2-CH2-CH2 on lysine (δ = 1.2- 1.8 ppm, peak 2, 3, 4) and calculated to be ~ 40 units. Number of CDM groups on the polymer was calculated by the peak of -CH3 (δ = 2.0 ppm, peak 6) and determined to be 15. FIG. 8 shows a size exclusion chromatography (SEC) of PEG-pLL(CDM) polymer. The result confirmed narrow molecular size distribution of the polymer.

[0078] FIGs. 9A-9B show a fluorescence cross-correlation spectroscopy of free IL- 15 ex and Nano-SA. FIG. 9A shows autocorrelation function curves of IL-15cx (left) and Nano-SA (right). FIG. 9B shows scattering plot of A647 and A488 fluorescence intensities in IL-15cx and Nano-SA samples. High cross-correlation between IL- 15 (A647 labelled) and IL-15Rα (A488 labelled) was found in both free IL-15cx and Nano-SA samples.

[0079] FIGs. 10A-10C show a reconstitution test of FRET-based formulations confirms the protection of Nano-S Aon the association between IL- 15 andIL-15Rα. FIG. 10 A is a schematic illustration of the experiment design. Free IL-15cx or Nano-SA were prepared with A647- labelled IL- 15 and A555 -labelled IL-15Rα. The samples were incubated with non-labelled IL- 15 or IL-15Rα for reconstitution. The FRET signal loss could reflect the reconstitution degree. FIG. 10B shows fluorescence emission spectra of the samples with excitation light at 555 nm. Intensities were normalized to the emission at 570 nm. . FIG. 10C shows FRET signals of the samples. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0080] FIG. 11 shows FCS measurement of IL-15cx and Nano-SA during incubation with murine plasma sample. IL-15 was labelled with A647 and IL-15Rα was labelled with A488. Cross-correlation function curves were plotted to reflect the association between IL-15 and IL- 15Rα.

[0081] FIG. 12A shows a schematic illustration showing the labelling strategy of the polymers used for the Al -Nano-SA formulation. The PEG-pLL(CDM) polymers were labelled by QSY- 21 quenchers through the condensation reaction between amines and NHS esters.

[0082] FIG. 12B shows characterization of AI-Nano-SA. Sample was incubated in buffers with different pH (7.4 or 6.5). The fluorescence emission intensities from A647 (Em 665 nm) and A488 (Em 510) were measured. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0083] FIG. 13 shows quantification of the FRET signal in vessels (white boxes in FIG. 3G) observed by IVCLSM.

[0084] FIG. 14 shows gating strategy of in vitro lymphocytes killing assay. Annexin V+ cells were specified as killed cells. FIG. 15 shows in vitro lymphocytes killing assay in YAC-1 cells. Data are plotted as the mean ± S.D., n = 4 independent samples.

[0085] FIG. 16 shows gating strategy of a flow cytometry experiment to define T cell, NK cell and NKT cell number and CD69 expression.

[0086] FIG. 17 shows flow cytometry analysis results determining the frequency and activation (CD69 expression) of CD4+ and CD8+ T cells in spleen, liver, and blood samples. Data are plotted as the mean ± S.D., n = 4 independent samples. Results were compared via one-way ANOVA.

[0087] FIG. 18 shows gating strategy of flow cytometry experiment to define the expression of Ki67 in T cells.

[0088] FIG. 19 shows Ki67 expression on CD4+ and CD8+ T cells in spleen, liver, and blood samples. Data are plotted as the mean ± S.D., n = 4 independent samples. Results were compared via one-way ANOVA.

[0089] FIG. 20 shows a heatmap showing the systemic immune response in blood, spleen, and liver samples. Values were converted to Z-scores and plotted.

[0090] FIG. 21 is a schematic illustration of an experiment schedule of efficacy and safety test of Nano-SA in a CT26 tumor model. Five daily Nano-SA or IL-15cx treatments (i.v., 3 μg IL- 15cx e.q.) were conducted in mice bearing CT26 tumors. Tumor growth was recorded to indicate the therapeutical efficacy. On Day 16 post-inoculation, the mice were sacrificed. Tumor and blood samples of the mice were collected for immune analysis and toxicity evaluation.

[0091] FIG. 22 shows cytokine concentrations in blood samples after the treatment in FIG. 16. Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0092] FIG. 23 shows cytokine concentrations in CT26 tumors after the treatment in FIG. 16. Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0093] FIG. 24 shows histological analysis of CT26 tumor sections. H&E staining and immuno staining against CD8 were performed. Scale bar = 1 mm. FIGs. 25A-25C show bodyweight changes of the mice bearing MC38 tumors during Nano-SA monotherapy treatment. FIG. 25A shows schematic illustration of the experiment schedule. FIG. 25B shows bodyweight change curves of each group. FIG. 25C shows bodyweight change of the animals on Day 2 post the first injection. Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0094] FIG. 26 shows serum IFN-γ concentration of the mice before (Day 5) and after (Day 10) treatment, n = 5 independent samples.

[0095] FIG. 27 show representative H&E staining images of organ sections from mice bearing MC38 tumors after different treatment. Scale bar 100 pm.

[0096] FIG. 28 shows a blood test measuring the organ damage-associated biomarkers after treatment. Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0097] FIG. 29 shows representative images of excised colons from mice bearing orthotopic MC38 tumors after different treatment. Scale bar = 1 cm.

[0098] FIGs. 30A-30B show antitumor efficacy of Nano-SA in mice bearing CT26 tumors. FIG. 30A shows tumor growth curves. FIG. 30B shows excised tumor weight on the termination point (Day 16 post inoculation). Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0099] FIGs. 31A-31B show antitumor efficacy of Nano-SA in mice bearing CT26 tumors. FIG. 31A shows tumor growth curves. FIG. 31B shows excised tumor weight on the termination point (Day 16 post inoculation). Data are plotted as the mean ± S.D., n = 5 independent samples. Results were compared via one-way ANOVA.

[0100] DETAILED DESCRIPTION

[0101] The present disclosure features protein-encapsulating polymeric complexes and methods of use thereof. In one aspect, a polymeric complex comprising an immunocomplex comprising a cytokine and a soluble cytokine-binding protein and a block copolymer is provided. The present disclosure provides, in part, a polymeric complex (e.g., a micelle) having the ability to form a polyion complex between the immunocomplex and the block copolymer and thereby encapsulate the immunocomplex through covalent bonding cleavable under given pH conditions, with the aim of releasing the loaded immunocomplex in a pH- dependent manner. In some embodiments, the immunocomplex delivery approach of using stimuli-responsive nanocarriers, as described in the present disclosure, enhances cytokine activity in target tissues in a tissue selective manner.

[0102] So that the disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0103] I. DEFINITIONS

[0104] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the present invention.

[0105] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. Also contemplated are embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. Also contemplated are embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0106] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0107] As a general matter, compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.

[0108] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. The terms “pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used interchangeably herein and refer to buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020).

[0109] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19, and Gould, Salt selection for basic drugs, International Journal of Pharmaceutics, 33 (1986) 201-217. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0110] As used herein, a “subject” to which administration is contemplated includes, but is not limited to, humans (e.g., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or a non-human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In certain embodiments, the subject is a human. In certain embodiments, the subject is a non- human animal. The terms “human,” “patient,” and “subject” are used interchangeably herein.

[0111] “Disease,” “disorder”, and “condition” are used interchangeably herein.

[0112] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes (a) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0113] The term “effective amount” as used herein refers to the amount of a compound, e.g., a compound or composition described herein, sufficient to elicit a beneficial or desired result or biological effect. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound or composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. For example, an effective amount may be an amount of a compound or composition sufficient to achieve one or more of the following: (i) modulate an activity of a cytokine signaling pathway, (ii) modulate an activity of a Janus-associated kinase (JAK) protein (e.g., a JAK3 protein), (iii) modulate an activity of a signal transducer and activator of transcription (STAT) protein (e.g., a STAT5 protein), (iii) modulate one or more transcriptional programs induced by STAT proteins, or (iv) treat or prevent a disease or condition associated with a cytokine signaling pathway, e.g., cancer, or a symptom or manifestation thereof.

[0114] As used herein, and unless otherwise specified, a “therapeutically effective amount” of a compound or composition is an amount sufficient to provide a therapeutic benefit in the treatment of a disease or condition, or to delay or minimize one or more symptoms associated with the disease, disorder or condition. As will be appreciated by those of ordinary skill in this art, a therapeutically effective amount of a compound or composition described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the age, weight, health, and condition of the subject. A therapeutically effective amount of a compound or composition can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route. A therapeutically effective amount of a compound or composition also includes an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the disease, disorder or condition. In addition, a therapeutically effective amount can encompass an amount of a compound or composition that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0115] At various places in the present specification, variable or parameters are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual subcombination of the members of such groups and ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0116] Throughout the description, where compounds or compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compounds or compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compounds or compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compounds or compositions of the present invention that consist of the recited components, and that there are processes and methods according to the present invention that consist of the recited processing steps.

[0117] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0118] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present invention, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present invention and / or in methods of the present invention, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and invention(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the invention(s) described and depicted herein.

[0119] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects should be understood to have the same meaning unless otherwise understood from the context.

[0120] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0121] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0122] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present invention and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present invention.

[0123] The claims encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group.

[0124] “Administer”, “administering”, or “administration”, as used herein, refer to implanting, absorbing, ingesting, injecting, or otherwise introducing an entity described herein (e.g., polymeric complex described herein), or providing the same to a subject.

[0125] “Cell,” as used herein, refers to an engineered cell or a cell that is not engineered. In an embodiment, a cell is an immortalized cell.

[0126] “Derived from”, as used herein with respect to a cell or cells, refers to a cell or cells obtained from tissue, a cell line, or other cells, which optionally are then cultured, passaged, immortalized, differentiated and / or induced, to produce the derived cell(s).

[0127] “Repeat unit” or “monomer” as used herein refers to the smallest unit (i.e., atom or group of atoms) which, when repeated, forms an oligomer or polymer.

[0128] “Polymer” as used herein refers to a chemical compound of relatively high molecular mass comprising a repeat unit of relatively low molecular mass. As used herein, polymer describes both naturally occurring and synthetic polymers.

[0129] “Block copolymer” as used herein refers to a polymer chain comprising more than one unique repeat unit. Each repeat unit is unique in the sense that it must have at least one chemical difference from other repeat units in the block. In some embodiments, a block copolymer as described herein has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more unique repeat units. In some embodiments, a block copolymer has 2, 3, 4, 5, 6, 7, 8, or 9 unique repeat units. In some embodiments, a block copolymer has 2, 3, 4, 5, 6, 7, or 8 unique repeat units. In some embodiments, a block copolymer has 2, 3, 4, 5, 6, or 7 unique repeat units. In some embodiments, a block copolymer has 2, 3, 4, 5, or 6 unique repeat units. In some embodiments, a block copolymer has 2, 3, 4, or 5 unique repeat units. In some embodiments, a block copolymer has 2, 3, or 4 unique repeat units. In some embodiments, a block copolymer has 2 or 3 unique repeat units. In some embodiments, a block copolymer has 2 unique repeat units.

[0130] “Charge regulator” as used herein refers to a compound that acts to covert the charge of a protein. For example, a charge regulator may act to convert the charge of a basic or neutral protein as a whole into that of an acidic protein. In some embodiments, a “charge regulator” is a compound represented by Formula (II). In some embodiments, a “charge regulator” is a compound represented by Formula (Il-a), Formula (Il-b), Formula (II-c), or Formula (Il-d). Details of how a charge regulator provided herein (e.g., a charge regulator of Formula (II)) accomplishes the aforementioned charge conversion are provided herein.

[0131] “Polyion complex” as used herein refers to a chemical bonding arrangement comprising more than one type of electrostatic bond (i.e., a bond between oppositely charged ions).

[0132] “Cytokine” as used herein refers to secreted proteins that act as messengers for regulation of the immune systems. In various embodiments, cytokines used in the complexes disclosed herein can be members of the interleukin (IL) family of cytokines. Interleukins activate receptors that signal through Signal Transducer and Activator of Transcription (STAT) transcription factors (e.g., STAT1, STAT3 and STAT5). Once activated, interleukin receptors can dimerize and bind Janus-associated kinases (JAKs) to induce JAK crossphosphorylation and downstream “JAK / STAT” signaling. Accordingly, induced cytokine expression can be utilized to induce receptor activity and thus cytokine signaling.

[0133] “Superagonisf ’ as used herein refers to any ligand that induces a maximum effect higher than endogenous ligand.

[0134] Chemical Definitions

[0135] Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75thEd., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March 's Advanced Organic Chemistry, 5thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rdEdition, Cambridge University Press, Cambridge, 1987.

[0136] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0137] Compounds described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereomers.

[0138] The terms “groups” and “radicals” can be considered interchangeable when used herein. When a range of values is listed, it is intended to encompass each value and subrange within the range. For example, “C1-C6alkyl” is intended to encompass, C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6alkyl.

[0139] As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 1 to 24 carbon atoms (“C1-C24alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C1-C12alkyl”), 1 to 10 carbon atoms (“C1-C12alkyl”), 1 to 8 carbon atoms (“C1-C8alkyl”), 1 to 6 carbon atoms (“C1-C6alkyl”), 1 to 5 carbon atoms (“C1-C5alkyl”), 1 to 4 carbon atoms (“C1-C4alkyl”), 1 to 3 carbon atoms (“C1-C3alkyl”), 1 to 2 carbon atoms (“C1-C2alkyl”), or 1 carbon atom (“C1alkyl”). In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C2-C6alkyl”). Examples of C1-C6alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tertbutyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents; e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C24alkenyl”). In some embodiments, an alkenyl group has 2 to 12 carbon atoms (“C2-C12alkenyl”), 2 to 10 carbon atoms (“C2-C10alkenyl”), 2 to 8 carbon atoms (“C2-C8alkenyl”), 2 to 6 carbon atoms (“C2-C6alkenyl”), 2 to 5 carbon atoms (“C2-C5alkenyl”), 2 to 4 carbon atoms (“C2-C4alkenyl”), 2 to 3 carbon atoms (“C2-C3alkenyl”), or 2 carbon atoms (“C2alkenyl”). The one or more carbon— carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4alkenyl groups include ethenyl (C2), 1— propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6alkenyl groups include the aforementioned C2-4alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Each instance of an alkenyl group may be independently optionally substituted, i. e. , unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g, for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0140] As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds (“C2-C24alkenyl”). In some embodiments, an alkynyl group has 2 to 12 carbon atoms (“C2-C10alkynyl”), 2 to 10 carbon atoms (“C2-C10alkynyl”), 2 to 8 carbon atoms (“C2-C8alkynyl”), 2 to 6 carbon atoms (“C2-C6alkynyl”), 2 to 5 carbon atoms (“C2-C5alkynyl”), 2 to 4 carbon atoms (“C2-C4alkynyl”), 2 to 3 carbon atoms (“C2-C3alkynyl”), or 2 carbon atoms (“C2alkynyl”). The one or more carbon-carbon triple bonds can be internal (such as in 2- butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each instance of an alkynyl group may be independently optionally substituted, i. e. , unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.

[0141] The terms "alkylene," “alkenylene,” or “alkynylene,” alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, or alkynyl, respectively. As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 71 electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14aryl”). In some embodiments, an aryl group has six ring carbon atoms (“C6aryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10aryl”; e.g., naphthyl such as 1 -naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14aryl”; e.g, anthracyl). An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the nonhydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, z. e. , unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.

[0142] As used herein, “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 107i electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.

[0143] In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1^4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more substituents.

[0144] Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6- bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0145] As used herein, “cycloalkyl” refers to a radical of a non-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“ C3-C8cycloalkyl”), 3 to 6 ring carbon atoms (“C3-C6cycloalkyl”), or 5 to 10 ring carbon atoms (“C5-C10cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4- Cv-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8cycloalkyl groups include, without limitation, the aforementioned C3-C6cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo [1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo [3.1.1]heptanyl (C7), and the like. Exemplary C3-C10cycloalkyl groups include, without limitation, the aforementioned C3-C8cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10), spiro [4.5] decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated.

[0146] “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more substituents.

[0147] “Heterocyclyl” as used herein refers to a radical of a 3- to 10-membered non- aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.

[0148] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1—4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0149] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrroly 1-2, 5-dione. Exemplary 5- membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl or thiomorpholinyl- 1,1 -dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0150] “Amino” as used herein refers to the radical -NR70R71, wherein R70and R71are each independently hydrogen, C1-C8alkyl, C3-C10cycloalkyl, C4-C10heterocyclyl, C6-C10aryl, and C5-C10heteroaryl. In some embodiments, amino refers to NH2.

[0151] As used herein, “halo” or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0152] As used herein, “hydroxy” refers to the radical -OH.

[0153] Alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any and all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.

[0154] Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group (such as an alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene or the carbon atom of a carbocyclyl, aryl, heterocyclyl or heteroaryl) are independently, for example, halogen; -(CH2)0-4R°; -(CH2)0-4OR°; -O-(CH2)0-4C(O)OR°; -(CH2)0-4CH(OR°)2;

[0155] -(CH2)0-4SR°; -(CH2)0-4Ph, which may be substituted with R°; -(CH2)0-40(CH2)0-1Ph, which may be substituted with R°; -CH=CHPh, which may be substituted with -NO2; -CN; -N3; - (CH2)0-4N(R°)2; -(CH2)0-4N(R°)C(O)R°; -N(R°)C(S)R°;

[0156] -(CH2)0-4N(R°) C(O)NR°2; -N(R°)C(S)NR°2; -(CH2)0-4N(R°)C(O)OR°; -N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)0-4C(O)R°; -C(S)R°; -(CH2)0-4C(O)O R°; -(CH2)0-4C(O)SR°; -(CH2)0-4C(O)OSiR°3; -(CH2)0-40C(O)R°; -OC(O)(CH2)0-4SR°-,

[0157] SC(S)SR°; -(CH2)0-4SC(O)R°; -(CH2)0-4C(O)NR°2; -C(S)NR°2; -C(S)SR°;

[0158] -(CH2)0-40C(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; -C(O)CH2C(O)R°;-C(NOR°)R°;

[0159] -(CH2)0-4SSR°; -(CH2)0-4S(O)2R°; -(CH2)0-4S(O)2OR°; -(CH2)0-4OS(O)2R°;

[0160] -S(O)2NR°2; -(CH2)0-4S(O)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2;

[0161] -P(O)2R°; -P(O)R°2; -OP(O)R°2; -OP(O)(OR°)2; -SiR3; -(C1-4straight or branched alkylene)O-N(R°)2; or -(C1-4straight or branched alkylene)C(O)O-N(R°)2, where each R° may be substituted as defined below and is independently hydrogen, C1-6alkyl, C1-6alkenyl, C1-6alkynyl, -CH2Ph, -0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.

[0162] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently, for example, deuterium, halogen, -(CH2)0-2R●, -(haloR●), -(CH2)0-2OH, -(CH2)0-2OR●, -(CH2)0-2CH(OR●)2; -O(haloR●), -CN, -N3, -(CH2)0-2C(O)R●, -(CH2)0-2C(O)OH, -(CH2)0-2C(O)OR●, -(CH2)0-2SR●, -(CH2)0-2SH, -(CH2)0-2NH2, -(CH2)0-2NHR●,

[0163] -(CH2)0-2NR●2, -NO2, -SiR●3, -OSiR●3, -C(O)SR●,

[0164] -(C1-4straight or branched alkylene)C(O)OR●, or -SSR●, where each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4alkyl, C1-4alkenyl, C1-4alkynyl, -CH2Ph,

[0165] -0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =O and =S.

[0166] Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: =O, =S, =NNR*2, =NNHC(O)R*, =NNHC(O)OR*, =NNHS(O)2R*, = NR*, =NOR*, -O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, where each independent occurrence of R* is selected from hydrogen, C1-6alkyl, C1-6alkenyl, C1-6alkynyl, and carbocyclyl, which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 hetero atoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: -O(C(R*)2)2-3O-, where each independent occurrence of R* is selected from hydrogen, C1-6alkyl, C1-6alkenyl, C1-6alkynyl, and carbocyclyl, which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.

[0167] Suitable substituents on the alkyl, alkenyl, alkynyl, or carbocyclyl group of R* include halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -C(O)OH, -C(O)OR●, -NH2, -NHR●, -NR●2, or -NO2, where each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-6alkyl, C1-6alkenyl, C1-6alkynyl, -CH2Ph, -0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include where each is independently hydrogen, C1-6alkyl, C1-6alkenyl, C1-6alkynyl, which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. In certain embodiments, it should be understood that a substitutable nitrogen can be substituted with four substituents (or have four bonds to less than four substituents) such that the nitrogen atom will be positively charged or its cation, i.e., N+. Such substitution of a substitutable nitrogen can be of a ring nitrogen, for example, a ring nitrogen of a heterocyclic group containing nitrogen as a ring atom. In particular embodiments, where one of the substituents of a cationic nitrogen atom is a hydroxyl group, the hydroxyl group can be deprotonated and represented by a negatively charged oxygen atom, i.e., O’. In such cases, a general formula for the substitution can be represented by RN+(O’)(R’)R”, where R, R’ and R” represent carbon or other atoms, groups and / or moieties to which the nitrogen atom is bound.

[0168] Suitable substituents on the alkyl, alkenyl, alkynyl, or carbocyclyl group of are independently, for example, halogen, -R●, -(haloR●), -OH, -OR●, -O(haloR●), -CN, -

[0169] C(O)OH, -C(O)OR●, -NH2, -NHR*, -NR●2, or -NO2, where each R●is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-6alkyl, C1-6alkenyl, C1-6alkynyl, -CH2Ph, -0(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocyclyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ringforming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0170] Polymers of Formula (I) described herein may comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including1H,2H (D or deuterium), and3H (T or tritium); C may be in any isotopic form, including12C,13C, and14C; O may be in any isotopic form, including16O and18O; and the like; F may be in any isotopic form, including18F and19F; and the like.

[0171] These and other exemplary substituents are described in more detail in the Detailed Description, Examples, and Claims. The invention is not intended to be limited in any manner by the above exemplary listing of substituents.

[0172] Certain polymers of Formula (I) described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain polymers of Formula (I) described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0173] The term “solvate” refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.

[0174] The term “hydrate” refers to a compound which is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R-x H2O, wherein R is the compound and wherein x is a number greater than 0.

[0175] The term “tautomer” as used herein refers to compounds that are interchangeable forms of a compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of n electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.

[0176] II. POLYMERIC COMPLEXES

[0177] In one aspect, the polymeric complex of the present disclosure is a protein-enclosing polymeric micellar complex (polyion complex: PIC), which comprises a cationic polymer (e.g., block copolymer, graft copolymer) and a protein (e.g., immunocomplex comprising: (a) a cytokine; and (b) a soluble cytokine-binding protein).

[0178] In some embodiments, the polymeric complexes of the present invention are characterized by the ability to self-assemble in a solution (e.g., an aqueous solution).

[0179] In one aspect, the present disclosure provides a polymeric complex comprising: (i) an immunocomplex comprising: (a) a cytokine; and (b) a soluble cytokine-binding protein; and (ii) a cationic polymer (e.g., block copolymer).

[0180] In some embodiments, the present disclosure provides a polymeric complex comprising a block copolymer, a protein, and a protein receptor. In some embodiments, the polymeric complex comprises a block copolymer, a cytokine (e.g., IL- 15), and a cytokine receptor (e.g., IL-15Rα).

[0181] In some embodiments, the polymeric complex comprises a block copolymer, a cytokine (e.g., IL- 15), and a cytokine receptor (e.g., IL-15Rα), wherein the block copolymer comprises a charge regulator, e.g., a charge regulator of Formula (II) (e.g., a charge regulator of Formula (Il-a), (Il-b), (II-c), or (II-d)).

[0182] (1) Cationic Polymer

[0183] In some embodiments, the cationic polymer has at least partially a polycation moiety. In some embodiments, the cationic polymer is a block copolymer or graft polymer having a polyethylene glycol (PEG) moiety and a polycation moiety.

[0184] In some embodiments, the polycation is a polypeptide having cationic groups in its side chains. As used herein, the term “cationic group” is intended to mean not only a group which is already cationic by being coordinated with hydrogen ions, but also a group which will be cationic when coordinated with hydrogen ions. In some embodiments, the polypeptide having cationic groups in its side chains includes those composed of known amino acids having a basic side chain (e.g., lysine, arginine, histidine) linked via peptide bonds, as well as those composed of various amino acids linked via peptide bonds, whose side chain (e.g., the side chain of aspartic acid or glutamic acid) is substituted to have a cationic group.

[0185] In some embodiments, the block copolymer comprises: (i) a group derived from a hydrophilic polymer; (ii) a group derived from a polypeptide having cationic groups in its side chains; and (iii) a group derived from a charge regulator.

[0186] In some embodiments, the hydrophilic polymer is soluble in aqueous solution between pH 5-9.

[0187] In some embodiments, the block copolymer comprises a group derived from polyethylene glycol.

[0188] In some embodiments, the block copolymer comprises a group derived from poly-L- lysine.

[0189] In some embodiments, the block copolymer comprises a group derived from a charge regulator, wherein the charge regulator is represented by a compound of Formula (II) (e.g., Formula (Il-a), Formula (Il-b), Formula (II-c), or Formula (II-d)).

[0190] In some embodiments, the block copolymer comprises: (i) a group derived from polyethylene glycol; (ii) a group derived from poly-lysine; and (iii) a group derived from a charge regulator (e.g., a charge regulator of Formula (II)).

[0191] In some embodiments, the block copolymer comprises: (i) a group derived from polyethylene glycol; (ii) a group derived from poly-L-lysine; and (iii) a group derived from a charge regulator (e.g., a charge regulator of Formula (II)).

[0192] In some embodiments, the block copolymer comprises: (i) a group derived from polyethylene glycol comprising 1-500 monomers; (ii) a group derived from poly-L-lysine comprising 1-500 monomers; and (iii) a group derived from a charge regulator (e.g., a charge regulator of Formula (II)).

[0193] In some embodiments, the block copolymer comprises: (i) a group derived from polyethylene glycol comprising 1-500 monomers; (ii) a group derived from poly-L-lysine comprising 1-500 monomers; and (iii) a group derived from a charge regulator (e.g., a charge regulator of Formula (II)). In some embodiments, the cationic polymer is a block copolymer represented by Formula (I):

[0194] In Formula (I), the block moiety whose number of repeating units (degree of polymerization) is n corresponds to the polyethylene glycol (PEG) moiety, while the block moiety composed collectively of submoieties whose number of repeating units is ml and m2, respectively (i.e., the moiety shown in brackets [ ] in Formula (I)) corresponds to the polycation moiety.

[0195] The symbol " / " indicates that (ml+m2) units of the respective monomer units shown on the left and right sides of this symbol may be in any sequence. For example, when a block moiety composed of monomer units A and B is represented by [-(A)a- / -(B)b-], the symbol " / " means that a units of A and b units of B, i.e., (a+b) units in total of the respective monomer units may be linked at random in any sequence (provided that all the monomer units A and B are linked in a linear fashion).

[0196] In Formula (I), R1and R2each independently represent a hydrogen atom, or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms, or a functional group such as an azide, an amine, maleimide, a ligand or a labeling agent.

[0197] Examples of the above linear or branched alkyl group containing 1 to 12 carbon atoms include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, a sec-butyl group, a tert-butyl group, a n-pentyl group, a n-hexyl group, a decyl group and an undecyl group, etc. Moreover, examples of substituents on the above alkyl group include an acetal-protected formyl group, a cyano group, a formyl group, a carboxyl group, an amino group, an alkoxycarbonyl group containing 1 to 6 carbon atoms, an acylamido group containing 2 to 7 carbon atoms, a siloxy group, a silylamino group, and a trialkylsiloxy group (each alkylsiloxy group independently contains 1 to 6 carbon atoms), etc. A ligand molecule refers to a compound used with the aim of targeting a certain biomolecule, and examples include an antibody, an aptamer, a protein, an amino acid, a low molecular compound, a monomer of a biological macromolecule and so on. Examples of a labeling agent include, but are not limited to, fluorescent labeling agents such as a rare earth fluorescent labeling agent, coumarin, dimethylaminosulfonyl benzoxadiazole (DBD), dansyl, nitrobenzoxadiazole (NBD), pyrene, fluorescein, a fluorescent protein and so on.

[0198] When the above substituent is an acetal-protected formyl group, this substituent can be converted into another substituent, i.e., a formyl group (or an aldehyde group; — CHO) upon hydrolysis under acidic mild conditions. Moreover, when the above substituent (particularly on R1) is a formyl group or is a carboxyl group or an amino group, for example, an antibody or a fragment thereof or other functional or targeting proteins may be linked via these groups.

[0199] In some embodiments, R1is C1- 12alkyl. In some embodiments, R1is methyl. In some embodiments, R2is hydrogen.

[0200] In Formula (I), R3is a group derived from a compound represented by Formula (II): wherein Raand Rbare each independently selected from the group consisting of: hydrogen, optionally substituted alkyl, alkenyl, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclyl, heterocyclic alkyl, hydroxy, alkoxy, and aryloxy group, or

[0201] Raand Rbare taken together with the carbon atoms to which they are attached to form an aromatic ring or a cycloalkyl ring, wherein the bond between the carbon atoms to which Raand Rbare attached may be a single bond or a double bond, wherein R3is covalently attached to the adjacent CH2.

[0202] In some embodiments, R3represents a direct bond to a compound represented by Formula (II).

[0203] In some embodiments, Raand Rbare selected from hydrogen and C1-C6alkyl.

[0204] In some embodiments, R3is selected from the group consisting of: wherein denotes the point of attachment.

[0205] In some embodiments, R3is: wherein denotes the point of attachment.

[0206] In Formula (I), L1represents NH, CO, a group represented by the following general formula (11):

[0207] — (CH2)p1— NH— (11)

[0208] (wherein p1 represents an integer of 1 to 6), or a group represented by the following general formula (12):

[0209] -L2a-(CH2)q1-L3a- (12)

[0210] (wherein L2arepresents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L3arepresents NH or CO, and q1 represents an integer of 1 to 6).

[0211] In some embodiments, L1is -(CH2)p1-NH-. In some embodiments, ql is 1.

[0212] In Formula (I), ml and m2 each independently represent an integer of 0 to 500 (provided that the sum of ml and m2 represents an integer of 10 to 500), and m3, m4 and m5 each independently represent an integer of 1 to 5.

[0213] In some embodiments, ml and m2 each independently represent an integer of 1 to 500 (provided that the sum of ml and m2 represents an integer of 10 to 500), and m3, m4 and m5 each independently represent an integer of 1 to 5. In some embodiments, ml represents an integer of 0 to 500 and m2 represents an integer greater than 0 up to 500, provided that the sum of ml and m2 represents an integer of 10 to 500.

[0214] In some embodiments, m3 is 1, 2, 3, 4, or 5. In some embodiments, m4 is 1, 2, 3, 4, or 5. In some embodiments, m5 is 1, 2, 3, 4, or 5.

[0215] In Formula (I), n represents the number of repeating units (degree of polymerization) in the PEG moiety, and more specifically represents an integer of 1 to 500. In some embodiments, n is an integer of 100 to 400. In some embodiments, n is an integer of 200 to 300.

[0216] In some embodiments, the molecular weight (Mn) of the cationic polymer represented by Formula (I) is from 23,000 to 45,000, or from 28,000 to 34,000.

[0217] With regard to the individual block moieties, in some embodiments, the PEG moiety has a molecular weight (Mw) of from 8,000 to 15,000, or from 10,000 to 12,000. In some embodiments, the polycation moiety as a whole has a molecular weight (Mn) of from 15,000 to 30,000, or from 18,000 to 22,000.

[0218] In some embodiments, wherein the block copolymer represented by Formula (I) is a block copolymer represented by Formula (la):

[0219] The cationic polymer represented by Formula (I) may be prepared in any manner. For example, a segment comprising R1and the block moiety of PEG chain (PEG segment) is synthesized in advance, and given monomers are sequentially polymerized to one end (opposite to R1) of this PEG segment, optionally followed by substituting or converting each side chain to contain a cationic group, or alternatively, the above PEG segment and a block moiety containing cationic groups in its side chains are synthesized in advance, which are then liked to each other. Procedures and conditions for each reaction in these preparation processes may be selected or determined as appropriate in consideration of standard processes.

[0220] In some embodiments, the protein (e.g., immunocomplex) is covalently bonded to the block copolymer represented by Formula (I).

[0221] The block copolymers of Formula (I) and Formula (la) are described in W02020262550, which is hereby incorporated by reference in its entirety for all purposes.

[0222] Charge regulators

[0223] In some embodiments, the block copolymer comprises a functional group capable of forming a covalent bond with a protein (e.g., a cytokine) under appropriate reaction conditions.

[0224] In some embodiments, the block copolymers comprise a group that acts as a charge regulator. In some embodiments, the charge regulator is a compound of Formula (II): wherein Raand Rbare each independently selected from the group consisting of: hydrogen, optionally substituted alkyl, alkenyl, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclyl, heterocyclic alkyl, hydroxy, alkoxy, and aryloxy group, or

[0225] Raand Rbare taken together with the carbon atoms to which they are attached to form an aromatic ring or a cycloalkyl ring, wherein the bond between the carbon atoms to which Raand Rbare attached may be a single bond or a double bond, wherein R3is covalently attached to the adjacent CH2.

[0226] In some embodiments, the compound represented by Formula (II) is at least one of compounds represented by Formulae (Ila) to (II g): In some embodiments, the compound represented by Formula (II) is a compound represented by Formula (Ila) or (lib):

[0227] The compound represented by Formula (II) acts to convert the charge of a basic or neutral protein as a whole into that of an acidic protein. In other words, the charge regulator of the present disclosure is deemed to cause overall charge conversion by controlling the amount of charge such that a protein whose overall charge is positive (+) or in neutral state is converted into a protein whose overall charge is negative (-). More specifically, the above overall charge conversion is accomplished as follows: the above compound represented by Formula (II) or a derivative thereof is bonded to an amino group (i.e., a positively charged group) contained in a protein, whereby the protein is negatively charged as a whole. For this purpose, this bonding is accomplished, for example, as follows: the above compound represented by Formula (II) is bonded (covalently bonded) to an amino group in a protein to form a structure as represented by the following formula (IF).

[0228] As to the above bonding, for example, when the above compound represented by Formula (II) is a compound represented by Formula (lIb) or (lie) shown above, the above structure represented by Formula (II’) formed after the bonding is as shown below.

[0229]

[0230] (2) Protein

[0231] In the PIC of the present disclosure, in some embodiments, the charge of the protein has been converted as a whole by a cationic polymer (e.g., a block co-polymer of Formula (I)) (i.e., a charge-conversional protein). In some embodiments, the overall charge of the protein has been converted from the overall charge of a basic or neutral protein (which is positive or in neutral state) into a negative charge, as in the case of the overall charge of an acidic protein. Such a protein whose overall charge has been converted into a negative charge can be regarded as an anionic substance (polyanion) when the protein is taken as a whole. Thus, upon electrostatic interaction with the polycation moiety in the above cationic polymer, such a charge-conversional protein can easily form a micellar complex which is inherently difficult to form with a basic or neutral protein.

[0232] Immunocomplex

[0233] In some embodiments, the protein is an immunocomplex comprising: (i) a cytokine; and (ii) a soluble cytokine-binding protein.

[0234] Exemplary cytokines

[0235] In one aspect, the present disclosure provides a polymeric complex comprising a protein, a protein receptor, and a block copolymer (e.g., a block copolymer of Formula (I)). In some embodiments, the polymeric complex comprises a cytokine, a cytokine receptor, and a block copolymer (e.g., a block copolymer of Formula (I)). In some embodiments, the cytokine has a neutral or positive charge at neutral pH. In some embodiments, the cytokine has a neutral charge at physiological pH. In some embodiments, the polymeric complex comprises a proinflammatory cytokine. In some embodiments, the polymeric complex comprises a proinflammatory cytokine shown in Table 1 A.

[0236] Table 1A. Exemplary human pro-inflammatory cytokines

[0237] In some embodiments the polymeric complex comprises a cytokine selected from: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

[0238] In some embodiments, the cytokine is human interleukin- 15 (IL-15; UniProt Accession No.: P40933). IL-15 is a pro-inflammatory cytokine that, inter alia, promotes the proliferation of immune cell, e.g., natural killer (NK) cells and T-cells.

[0239] In some embodiments, the IL- 15 is a human IL-15. In some embodiments, the IL- 15 is “IL-15 isoform 1 (UniProt Accession No.: P40933-1). In some embodiments, the IL-15 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-15 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-15 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-15 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-15 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL-15 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the IL- 15 comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0240] In some embodiments, the IL-15 is IL-15 isoform 2 (UniProt Accession No.: P40933- 2). In some embodiments, the IL-15 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-15 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-15 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-15 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the IL-15 comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0241] In some embodiments, the IL- 15 is the mature IL- 15 protein. In some embodiments, the IL- 15 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL-15 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 257. In some embodiments, the IL- 15 comprises the amino acid sequence set forth in SEQ ID NO: 257.

[0242] In some embodiments, the polymeric complex comprises IL-15.

[0243] In some embodiments, the polymeric complex comprises to an ortholog of IL-15. In some embodiments, the polymeric complex comprises an IL- 15 ortholog listed in Table IB. In some embodiments, the polymeric complex comprises a mature protein of an IL- 15 ortholog listed in Table IB.

[0244] Table IB. Exemplary non-human IL-15 orthologs

[0245] In some embodiments, the polymeric complex comprises an IL- 15 ortholog. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a amino acid sequence selected from any one of: SEQ ID NOs: 6-243 and 277- 286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 85% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL-15 ortholog comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL-15 ortholog comprises an amino acid sequence at least 91% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 92% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL-15 ortholog comprises an amino acid sequence at least 93% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortho log comprises an amino acid sequence at least 94% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL-15 ortholog comprises an amino acid sequence at least 96% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 97% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 98% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 99.5% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6- 243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence at least 99.9% identical to an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286. In some embodiments, the IL- 15 ortholog comprises an amino acid sequence selected from any one of SEQ ID NOs: 6-243 and 277-286.

[0246] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (III) (SEQ ID NO: 258): MX1X2X3KPX4X5X6X7X8SX9X10X11YX12CX13LLX14X15X16X17X18X19EAX20, wherein X1is R or K; X2is I, G, or M; X3is S, L, H, or E; X4is H, Y, R, S, or N; X5is L or M; X6is R or G; X7is S, I, or N; X8is I, V, S, P, or T; X9is I or F; X10is Q, H, or S; X11is C or S; X12is L or V; X13is L or F; X14is N, K, or H; X15is S or I; X16is H, R, or Q; X17is F, L, or I; X18is L or I; X19is T, A, or N; and X20is G or C. In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (IILa) (SEQ ID NO: 259): MRX2X3KPX4LX6X7X8SX9QX11YX12CX13LLX14X15HX17LX19EAG wherein X2is I, G, or M; X3is S, L, H, or E; X4is H, Y, R, S, or N; X6is R or G; X7is S, I, or N; X8is I, V, S, P, or T; X9is I or F; X11is C or S; X12is L or V; X13is L or F; X14is N, K, or H; X15is S or I; X17is F, L, or I; and X19is T, A, or N.

[0247] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (III-b) (SEQ ID NO: 260): MRIX3KPX4LRX7X8SIQCYX12CLLLX14X15HFLX19EAG wherein X3is S, L, H, or E; X4is

[0248] H, Y, R, S, or N; X7is S, I, or N; X8is I, V, S, P, or T; X12is L or V; X14is N, K, or H; X15is S or I; and X19is T, A, or N.

[0249] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (III-c) (SEQ ID NO: 261): MRIX3KPX4LRSX8SIQCYX12CLLLNSHFLTEAG wherein X3is S, L, H, or E; X4is H, Y, R, S, or N; X8is I, V, S, P, or T; and X12is L or V.

[0250] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (Ill-d) (SEQ ID NO: 262): MRIX3KPX4LRSX8SIQCYX12CLLLNSHFLTEAG wherein X3is S, L, H, or E; X4is H, Y, R, S, or N; X8is I, V, S, P, or T; and X12is L or V.

[0251] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (IV): Y1Y2Y3FY4Y5Y6Y7Y8Y9Y10Y11Y12PY13Y14EY15wherein Y1is I or M; Y2is H, R, or P; Y3is V or I; Y4is I, V, or L; Y5is L, F, or W; Y6is G or S; Y7is C, R, or S; Y8is F, V,

[0252] I, or T; Y9is S, G, or N; Y10is A, V, or T; Y11is G, A, or S; Y12is L or I; Y13is K, R, E, or N; Y14is T or I; and Y15is A or S.

[0253] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (IV-a) (SEQ ID NO: 263): IY2VFY4Y5Y6CY8SY10Y11Y12PY13TEA wherein Y2is H, R, or P; Y4is I, V, or L; Y5is L, F, or W; Y6is G or S; Y8is F, V, I, or T; Y10is A, V, or T; Y11is G, A, or S; Y12is L or I; and Y13is K, R, E, or N.

[0254] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (IV-b) (SEQ ID NO: 264): IY2VFY4Y5Y6CY8SAY11LPY13TEA wherein Y2is H, R, or P; Y4is I, V, or L; Y5is L, F, or W; Y6is G or S; Y8is F, V, I, or T; Y11is G, A, or S; and Y13is K, R, E, or N.

[0255] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (IV-d) (SEQ ID NO: 265): IHVFILGCY8SAGLPKTE A wherein Y8is F, V, I, or T.

[0256] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (V) (SEQ ID NO: 266): Z1WZ2Z3VZ4Z5DLZ6Z7Z8Z9Z10Z11IZ12wherein Z1is N, I, S, Q, or T; Z2is V, Q, E, C, N, H, D, or K; Z3is N, D, H, F, or Y; Z4is I, R, or M; Z5is S, Q, K, R, or H; Z6is K, R, Q, N, or E; Z7is K, R, T, M, or A; Z8is I or F; Z9is E, Q, K or D; Z10is D, Q, T, N, Y, H, G, S, or K; Z11is L or I; and Z12is Q or K.

[0257] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (V-a) (SEQ ID NO: 267): Z1WZ2Z3VZ4Z5DLZ6Z7Z8Z9Z10Z11IZ12wherein Z1is N, I, S, Q, or T; Z2is V, Q, E, C, N, H, D, or K; Z3is N, D, H, F, or Y; Z4is I, R, or M; Z5is S, Q, K, R, or H; Z6is K, R, Q, N, or E; Z7is K, R, T, M, or A; Z8is I or F; Z9is E, Q, K or D; Z10is D, Q, T, N, Y, H, G, S, or K; Z11is L or I; and Z12is Q or K.

[0258] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of NWVNVISDLKKIEDLIQ (SEQ ID NO: 268).

[0259] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (V-b) (SEQ ID NO: 269): NWZ2Z3VISDLKKIEDLIQ wherein Z2is V, Q, E, C, N, H, D, or K; and Z3is N, D, H, F, or Y.

[0260] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (VI): TLYTESD (SEQ ID NO: 270).

[0261] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (VII): HPSCK (SEQ ID NO: 271).

[0262] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (VIII):

[0263] VTAMKCFLLELQVISLES (SEQ ID NO: 272).

[0264] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (IX):

[0265] GDASIHDTVENLIILANN (SEQ ID NO: 273).

[0266] In some embodiments, the cytokine (e.g., IL-15) comprises an amino acid sequence of Formula (X):

[0267] ECEELEEKN (SEQ ID NO: 274).

[0268] In some embodiments, the cytokine (e.g., IL- 15) comprises an amino acid sequence of Formula (XI):

[0269] IKEFLQSFVHIVQMFIN (SEQ ID NO: 275).

[0270] Free IL- 15 is generally characterized by a weak ability to agonize effector cells (e.g., T cells or NK cells). However, binding IL-15 with IL-15 receptor domain a (IL-15Rα; Accession No.: Q13261) enhances IL-15 signaling by trans-presentation (see, e.g., Stonier, Spencer W., and Kimberly S. Schluns. "Trans-presentation: a novel mechanism regulating IL- 15 delivery and responses." Immunology letters 127.2 (2010): 85-92.) IL-15 binds with the protein IL-15Rα to form a complex called an IL- 15 superagonist (i.e., the complex is a better agonist than free IL- 15 alone). IL- 15 superagonists have been investigated in clinical trials as a possible treatment for advanced cancers.

[0271] In some embodiments, a complex comprises an IL-15Rα In some embodiments, IL- 15Rα refers to IL-15Rα isoform 1 (UniProt Accession No.: Q13261-1), IL-15 isoform 2 (UniProt Accession No.: Q13261-2), IL-15 isoform 3 (UniProt Accession No.: Q13261-3), IL-15 isoform 4 (UniProt Accession No.: Q13261-4), IL-15 isoform 5 (UniProt Accession No.: Q13261-5), IL-15 isoform 6 (UniProt Accession No.: Q13261-6), IL-15 isoform 7 (UniProt Accession No.: Q1326L7), IL-15 isoform 8 (UniProt Accession No.: Q13261-8), or IL-15 isoform 9 (UniProt Accession No.: Q13261-9).

[0272] In some embodiments, IL-15Rα refers to IL-15Rα isoform 1, IL-15Rα isoform 2, IL- 15Rα isoform 3, IL-15Rα isoform 4, or IL-15Rα isoform 9.

[0273] In some embodiments, IL-15Rα does not refer to IL- 15 isoform 5, IL- 15 isoform 6, IL- 15 isoform 7, and IL- 15 isoform 8.

[0274] Table 1C.

[0275] Signal peptides in the table above are identified in bold.

[0276] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to a amino acid sequence selected from any one of: SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 85% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 93% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 97% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.9% identical to an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence selected from any one of SEQ ID NOs: 244-252. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0277] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 244. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0278] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL-15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0279] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL-15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 246. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0280] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 247. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0281] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 248. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO:

[0282] 248. In some embodiments, the IL-15 receptor does not comprise a signal peptide.

[0283] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 249. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO:

[0284] 249. In some embodiments, the IL-15 receptor does not comprise a signal peptide. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 250. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0285] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 251. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0286] In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL-15 receptor comprises the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the IL- 15 receptor does not comprise a signal peptide.

[0287] In some embodiments, IL-15Rα refers to an ortholog of IL-15Rα.

[0288] MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYIC NSGFKRKAGTSSLTECVLNKATNVAHRDPALVHQRPAPPSTVTTAGVTPQPESLSPSG KEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELT ASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAM EALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 276).

[0289] In some embodiments, the cytokine is human interleukin-2 (IL-2; UniProt Accession No.: P60568). IL-2 is a pro-inflammatory cytokine that, inter alia, promotes the proliferation of immune cell, e.g., natural killer (NK) cells and T-cell.

[0290] In some embodiments, the IL-2 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 1 . In some embodiments, the IL-2 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the IL-2 does not comprise a signal peptide.

[0291] In some embodiments, a complex comprises an IL-2 receptor. In some embodiments the IL-2 receptor comprises IL-2Rα (UniProt Accession No. P01589).

[0292] IL-2Rα amino acid sequence:

[0293] MDSYLLMWGLLTFIMVPGCQAELCDDDPPEIPHATFKAMAYKEGTM LNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQV TPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHF VVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTGE METSQFPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTT EYQVAVAGCVFLLSVLLLSGLTQRRQRKSRRTI (SEQ ID NO: 253)

[0294] (signal peptide indicated in bold)

[0295] IL-2Rα sushi domain 1 amino acid sequence:

[0296] ELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTG

[0297] NSSHSSWDNQCQCTS (SEQ ID NO: 254)

[0298] IL-2Rα sushi domain 2 amino acid sequence:

[0299] GHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCK MTHGKTRWTQPQLICTG (SEQ ID NO: 255)

[0300] In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 253. In some embodiments, the IL-2 receptor comprises the amino acid sequence set forth in SEQ ID NO:

[0301] 253. In some embodiments, the IL-2 receptor does not comprise a signal peptide.

[0302] In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 93 % identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 254. In some embodiments, the IL-2 receptor comprises the amino acid sequence set forth in SEQ ID NO:

[0303] 254. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 255. In some embodiments, the IL-2 receptor comprises the amino acid sequence set forth in SEQ ID NO: 255.

[0304] In some embodiments, the cytokine is human interleukin-4 (IL-4; UniProt Accession No.: P05112). IL-4 is a pro-inflammatory cytokine that, inter alia, promotes activity of B cells, lymphocytes, monocytes, and macrophages. In some embodiments, the IL-4 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 comprises the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the IL-4 does not comprise a signal peptide.

[0305] In some embodiments, a complex comprises an IL-4 receptor. In some embodiments the IL-4 receptor comprises IL-4Rα (UniProt Accession No. P24394).

[0306] IL-4Rα amino acid sequence:

[0307] MGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNG PTNCSTELRLLYQLVFLLSEAHTCIPENNGGAGCVCHLLMDDVVSADNYTLD LWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNPYPPDNYLY NHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCY NTTWSEWSPSTKWHNSYREPFEQHLLLGVSVSCIVILAVCLLCYVSITKIKKE WWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFL EHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFE APVECEEEEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENG GFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPA SPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEP EMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVH AVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKP FQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVED MPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMA SPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSF HPAPGNAQ S S SQTPKIVNFVS VGPTYMRVS

[0308] (SEQ ID NO: 256) (signal peptide indicated in bold)

[0309] In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor comprises the amino acid sequence set forth in SEQ ID NO: 256. In some embodiments, the IL-4 receptor does not comprise a signal peptide.

[0310] In some embodiments, the cytokine is human interleukin-2 (IL-7; UniProt Accession No.: P13232). IL-7 is a pro-inflammatory cytokine that, inter alia, promotes the proliferation of immune cell, e.g., natural killer (NK) cells and T-cell.

[0311] In some embodiments, the IL-7 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the IL-7 does not comprise a signal peptide.

[0312] In some embodiments, a complex comprises an anti-IL-7 antibody.

[0313] Properties of Particles

[0314] It is generally understood that cytokines can have a short-half life in vivo and that maintaining a high dose is necessary to maintain its effect. However, systemic administration of a proinflammatory cytokine at high doses (e.g., by intravenous injection) is unfeasible due to adverse, off-target effects. Therefore, there is an unmet need of providing proinflammatory cytokines directly to a target location for therapeutic timescales. There is a particular need to deliver superagonist to a cold-tumor microenvironment at doses sufficient to revive or stimulate immune cell activation and infiltration.

[0315] The present disclosure describes, inter alia, polymeric complexes comprising a protein, a protein receptor, and a block copolymer. As described herein, the enclosure of a protein in a block copolymer of Formula (I) or Formula (la) protects the protein from the harsh in vivo environment. Moreover, the enclosure of a protein and its cognate receptor in a block copolymer (e.g., a block copolymer of Formula (I) or Formula (la)) may impart a number of other advantageous properties, including increased blood half-life, increased tumor uptake, improved biodistribution, an increase or decrease in cytokine levels, and / or increased survival in a subject.

[0316] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), and a protein.

[0317] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), and a cytokine.

[0318] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), and a proinflammatory cytokine.

[0319] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), and a proinflammatory cytokine selected from: IL- 1, IL-6, IL-8, IL-11, IL-15, IL-17, IL-18, IFN-α, IFN-β, IFN-γ, TNF-α, and TNF-β. In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), IL-15, and optionally farther comprising IL-15Rα.

[0320] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), IL-15, and farther comprising IL-15Rα, optionally wherein IL- 15 or IL-15Rα proteins further comprise a detection tag.

[0321] In some embodiments of the invention, the particle comprises a block copolymer according to Formula (I) or Formula (la), IL-15, and IL-15Rα.

[0322] Size

[0323] The polymeric complexes (e.g., micelles) of the present application may be characterized by their size. In some embodiments, the micelles may be characterized by their longest length in a particular dimension (i.e., a longest linear dimension). In some embodiments, the micelles are characterized by their longest linear dimension. In some embodiments, the micelles are characterized by their diameter.

[0324] The measurement of the size of the micelles may be performed by any method known in the art. In some embodiments, the size of the micelles is measured by a method selected from: dynamic light scattering (DLS), gel permeation chromatography (GPC), and transmission electron microscopy (TEM). In some embodiments, the size of the micelles is measured by DLS. In some embodiments, the size of the micelles is measured by GPC. In some embodiments, the size of the micelles is measured by TEM.

[0325] In some embodiments, the size of the micelles is measured by DLS. In some embodiments, the size of the micelles is reported as a hydrodynamic diameter. As will be understood by those having ordinary skill in the art, a hydrodynamic diameter will refer to an average hydrodynamic diameter.

[0326] The hydrodynamic diameter of micelles of the present invention may refer to a range of values. In some embodiments, the hydrodynamic diameter refers to a range of about 10 nm to 100 nm, 10 nm to 90 nm, 10 nm to 80 nm, 10 nm to 70 nm, 10 nm to 60 nm, 10 nm to 50 nm, 10 nm to 40 nm, 10 nm to 30 nm, 10 nm to 20 nm, 20 nm to 100 nm, 20 nm to 90 nm, 20 nm to 80 nm, 20 nm to 70 nm, 20 nm to 60 nm, 20 nm to 50 nm, 20 nm to 40 nm, 20 nm to 30 nm, 30 to 100 nm, 30 to 90 nm, 30 to 80 nm, 30 to 70 nm, 30 to 60 nm, 30 to 50 nm, 30 to 40 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm to 60 nm, 40 nm to 50 nm, 50 nm to 100 nm, 50 nm to 90 nm, 50 nm to 80 nm, 50 nm to 70 nm, 50 nm to 60 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, 60 nm to 70 nm, 70 nm to 100 nm, 70 nm to 90 nm, 70 nm to 80 nm, 80 nm to 100 nm, 80 nm to 90 nm, or 90 nm to 100 nm. In some embodiments, the hydrodynamic diameter of micelles of the present invention refers to a range of less than about 10 nm to 100 nm, 10 nm to 90 nm, 10 nm to 80 nm, 10 nm to 70 nm, 10 nm to 60 nm, 10 nm to 50 nm, 10 nm to 40 nm, 10 nm to 30 nm, 10 nm to 20 nm, 20 nm to 100 nm, 20 nm to 90 nm, 20 nm to 80 nm, 20 nm to 70 nm, 20 nm to 60 nm, 20 nm to 50 nm, 20 nm to 40 nm, 20 nm to 30 nm, 30 to 100 nm, 30 to 90 nm, 30 to 80 nm, 30 to 70 nm, 30 to 60 nm, 30 to 50 nm, 30 to 40 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm to 60 nm, 40 nm to 50 nm, 50 nm to 100 nm, 50 nm to 90 nm, 50 nm to 80 nm, 50 nm to 70 nm, 50 nm to 60 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, 60 nm to 70 nm, 70 nm to 100 nm, 70 nm to 90 nm, 70 nm to 80 nm, 80 nm to 100 nm, 80 nm to 90 nm, or 90 nm to 100 nm.

[0327] In some embodiments, the hydrodynamic diameter of micelles of the present invention refers to a range of greater than about 10 nm to 100 nm, 10 nm to 90 nm, 10 nm to 80 nm, 10 nm to 70 nm, 10 nm to 60 nm, 10 nm to 50 nm, 10 nm to 40 nm, 10 nm to 30 nm, 10 nm to 20 nm, 20 nm to 100 nm, 20 nm to 90 nm, 20 nm to 80 nm, 20 nm to 70 nm, 20 nm to 60 nm, 20 nm to 50 nm, 20 nm to 40 nm, 20 nm to 30 nm, 30 to 100 nm, 30 to 90 nm, 30 to 80 nm, 30 to 70 nm, 30 to 60 nm, 30 to 50 nm, 30 to 40 nm, 40 nm to 100 nm, 40 nm to 90 nm, 40 nm to 80 nm, 40 nm to 70 nm, 40 nm to 60 nm, 40 nm to 50 nm, 50 nm to 100 nm, 50 nm to 90 nm, 50 nm to 80 nm, 50 nm to 70 nm, 50 nm to 60 nm, 60 nm to 100 nm, 60 nm to 90 nm, 60 nm to 80 nm, 60 nm to 70 nm, 70 nm to 100 nm, 70 nm to 90 nm, 70 nm to 80 nm, 80 nm to 100 nm, 80 nm to 90 nm, or 90 nm to 100 nm.

[0328] In some embodiments, the hydrodynamic diameter of micelles of the present invention refers to a range of about 30 nm to 40 nm.

[0329] In some embodiments, the hydrodynamic diameter of micelles of the present invention is about 30 nm.

[0330] In some embodiments, the hydrodynamic diameter of micelles of the present invention is greater than about 30 nm.

[0331] In some embodiments, the hydrodynamic diameter of micelles of the present invention is about 35 nm.

[0332] In some embodiments, the hydrodynamic diameter of micelles of the present invention is about 40 nm.

[0333] In some embodiments, the hydrodynamic diameter of micelles of the present invention is less than about 40 nm.

[0334] Diffusion Coefficient The micelles of the present invention may be characterized by their diffusion coefficient (e.g., as compared to a reference standard).

[0335] The measurement of the diffusion coefficient of the micelles may be performed by any method known in the art. In some embodiments, the size of the micelles is measured by fluorescence correlation spectroscopy (FCS).

[0336] The diffusion coefficient of micelles of the present invention may refer to a range of diffusion coefficients. In some embodiments, the diffusion coefficient of micelles of the present invention refers to a range of about 50 cm2s-1to 10 cm2s-1, 50 cm2s-1to 20 cm2s-1, 50 cm2s-1to 30 cm2s-1, 50 cm2s4to 40 cm2s-1, 40 cm2s-1to 10 cm2s-1, 40 cm2s-1to 20 cm2s-1, 40 cm2s-1to 30 cm2s-1, 30 cm2s-1to 10 cm2s-1, 30 cm2s-1to 20 cm2s-1, or 20 cm2s-1to 10 cm2s-1.

[0337] In some embodiments, the diffusion coefficient of micelles of the present invention refers to a range of less than about 50 cm2s-1to 10 cm2s-1, 50 cm2s-1to 20 cm2s-1, 50 cm2s-1to 30 cm2s-1, 50 cm2s-1to 40 cm2s-1, 40 cm2s-1to 10 cm2s-1, 40 cm2s-1to 20 cm2s-1, 40 cm2s-1to 30 cm2s-1, 30 cm2s-1to 10 cm2s-1, 30 cm2s-1to 20 cm2s-1, or 20 cm2s-1to 10 cm2s-1.

[0338] In some embodiments, the diffusion coefficient of micelles of the present invention refers to a range of greater than about 50 cm2s-1to 10 cm2s-1, 50 cm2s-1to 20 cm2s-1, 50 cm2s-1to 30 cm2s-1, 50 cm2s-1to 40 cm2s-1, 40 cm2s-1to 10 cm2s-1, 40 cm2s-1to 20 cm2s-1, 40 cm2s-1to 30 cm2s-1, 30 cm2s-1to 10 cm2s-1, 30 cm2s-1to 20 cm2s-1, or 20 cm2s-1to 10 cm 2 s-1.

[0339] In some embodiments, the diffusion coefficient of micelles of the present invention is about 20 cm2s-1to 10 cm2s-1.

[0340] In some embodiments, the diffusion coefficient of micelles of the present invention is less than 20 cm2s-1.

[0341] In some embodiments, the diffusion coefficient of micelles of the present invention is greater than 10 cm2s-1.

[0342] Detection Tags

[0343] In some embodiments, the micelles of the present invention are characterized by the presence of a detection tag.

[0344] Stability

[0345] In some embodiments, the micelles of the present invention are characterized by the ability to protect a cargo or payload (e.g., a protein) from release in certain conditions. Blood circulation In some embodiments, the micelles of the present invention are characterized by the ability to increase the circulation time of a cargo or payload (e.g., as compared to the unencapsulated cargo or payload).

[0346] Tumor uptake

[0347] In some embodiments, the micelles of the present invention are characterized by the ability to increase the intratumoral uptake of a cargo or payload (e.g., as compared to the unencapsulated cargo or payload).

[0348] Minimization of off-target effects

[0349] In some embodiments, the micelles of the present invention are characterized by the ability to minimize off-target effects of a cargo or payload (e.g., as compared to unencapsulated cargo).

[0350] In some embodiments, the micelles of the present invention are characterized by the ability to minimize the increase in the levels of a cytokine selected from: IFN-y, IL-6, IL- 10, TGF-b, and CRE (e.g., as compared to unencapsulated cargo).

[0351] In some embodiments, the micelles of the present invention are characterized by the ability to minimize the increase in the levels of an organ damage marker selected from: ALT, TP, and BUN.

[0352] CD8+ infiltration

[0353] The micelles of the present invention are characterized by the ability to increase the penetration of CD8+ cells into the tumor volume (e.g., as compared to free protein).

[0354] III. PHARMACEUTICAL COMPOSITIONS AND ROUTES OF ADMINISTRATION

[0355] The polymeric complexes provided in accordance with the present disclosure, e.g., a polymeric complex comprising (i) an immunocomplex comprising (a) a cytokine; and (b) soluble cytokine-binding protein; and (ii) a block copolymer represented by Formula (I)) are usually administered in the form of pharmaceutical compositions. Provided herein are pharmaceutical compositions that contain, as the active ingredient, a polymeric complex described herein, and one or more pharmaceutically acceptable excipients, carriers, including inert solid diluents and fillers, diluents, including sterile aqueous solution and various organic solvents, permeation enhancers, solubilizers and adjuvants. The pharmaceutical compositions may be administered alone or in combination with other therapeutic agents. Such compositions are prepared in a manner well known in the pharmaceutical art (see, e.g. , Adeboye Adejare, Remington: The Science and Practice of Pharmacy (23rd ed. 2020)).

[0356] In certain embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such 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, 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; and / or excipients (see Adeboye Adejare (2020) supra}.

[0357] The pharmaceutical compositions may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities.

[0358] One mode for administration is parenteral, particularly by injection and can be administered locally, such as intramuscularly, intraarticularly, subcutaneously, intradermally, in addition to conventional systemic administration such as intravenous or intraarterial administration. The forms in which the polymeric complexes described herein may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, com oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. Aqueous solutions in saline are also conventionally used for injection, but less preferred in the context of the present invention. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[0359] Sterile injectable solutions are prepared by incorporating a polymeric complex according to the present disclosure in the required amount in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0360] The compositions are preferably formulated in a unit dosage form. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient (e.g., a tablet, capsule, ampoule). The polymeric complexes are generally administered in a pharmaceutically effective amount. For parenteral administration, from 0.1 to 700 mg (e.g., from 0.1 mg to 300 mg, from 0.1 mg to 100 mg, from 0.1 mg to 10 mg) of a polymeric complexes described herein. The reference to polymeric complex includes the polymeric complex either alone or in combination with a pharmaceutically acceptable salt. It will be understood, however, that the amount of the polymeric complex actually administered usually will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual polymeric complex administered and its relative activity, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like. IV. METHODS OF TREATMENT

[0361] In various embodiments, the invention provides methods of treating an immune- related condition (e.g., a cancer) in a subject in need thereof comprising administering to the individual an effective amount of a composition comprising a polymeric complex disclosed herein. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, melanoma, breast cancer, lung cancer, pancreatic cancer, brain cancer, head and neck cancer, liver cancer, stomach cancer, and mesothelioma. In some embodiments, the cancer is a colorectal cancer. In some embodiments, the cancer is a melanoma. In a preferred embodiment, the tumor type is a “hot” inflamed tumor. “Hot” inflamed tumors are defined as tumors with a high infiltration of antitumor immune cells or immunoisolated tumors where the antitumor immune cells are located in the periphery of the tumor. Further details on immunological classification of tumors is provided in Ren et al. Front Immunol. 2022; 13:790113.

[0362] In some embodiments, the method of treating cancer further comprises administering a checkpoint inhibitor to the subject. In some embodiments, the checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof. In some embodiments, the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

[0363] In some embodiments, the present disclosure provides a method of modulating an immune response in a subject receiving an immunotherapy regimen comprising: (i) administering a complex of the present disclosure to the subject; (ii) measuring or having measured a biological property of the subject; (iii) changing the course of the immunotherapy regimen based on the measurement of the biological property; thereby modulating the immune response in the subject. In some embodiments, the immune complex further comprises a detectable agent (e.g., a fluorescent agent or a radioimaging agent).

[0364] In various embodiments, administration of a polymeric complex of the present disclosure provides improved therapeutic results compared to a reference standard. In some embodiments, the reference standard is an immunocomplex not comprising the block copolymer. In some embodiments, the reference standard is an immune complex that is not formulated in a nanoparticle (e.g., a lipid nanoparticle). In some embodiments, the reference standard is a naked immunocomplex. In some embodiments, administration of the complex results in reduced or substantially no off-target toxicity in the subject compared to a reference standard. In some embodiments the off-target toxicity is measured by serum cytokine levels; serum ALT, TP, BUN, or CRE levels; and or body weight of the subject. In some embodiments, the serum cytokine levels are reduced by 2-fold compared to a reference standard. In some embodiments, the serum ALT, TP, BUN, or CRE levels are reduced by half compared to a reference standard. Serum cytokine and / or serum levels of ALT, TP, BUN, or CRE can be measured by any suitable method known in the art (e.g., ELISA or dry chemistry). In some embodiments, the body weight of the subject is increased by 10% compared to a reference standard. In some embodiments, the reference standard is an IL-15 / IL-15Rα immunocomplex without a block copolymer.

[0365] In some embodiments, administration of the complex results in reduced or substantially no increase in splenic weight in the subject compared to a reference standard.

[0366] V. METHODS OF DELIVERING IMMUNOCOMPLEXES

[0367] In various embodiments, the present disclosure provides methods of delivering an immunocomplex (e.g., a superagonist) to a cell. In some embodiments the pH of a region surrounding the cell permits release of cytokine and a cytokine receptor or cytokine-binding fragment thereof. In some embodiments, the pH of the region surrounding the cell is less than about 7.0. In some embodiments, the complex exhibits reduced or substantially no disassembly of the immunocomplex compared to a reference standard. In some embodiments, disassembly of the complex is reduced by pH higher than 7.0.

[0368] EXAMPLES

[0369] In order that the disclosure described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the particles, compositions, and methods provided herein and are not to be construed in any way as limiting their scope.

[0370] Labelling of Proteins and Polymers General Method

[0371] Proteins and polymers were labelled with different fluorescence dyes or quenchers. All labelling was conducted via a condensation reaction between the succinimidyl ester and an amine under the condition suggested by the manufacturer of the reagents. For proteins, the reactions were processed in NaHCO3buffer (50 mM, pH 8.4). For polymers, the reaction was processed in acetate buffer (50 mM, pH 5.5). All labelled products were purified by gel filtration through PD-10 columns filled with Sephadex G-25 gel to remove the unreacted labelling reagents.

[0372] Establishment of tumor models

[0373] All tumor models were established in female mice at 6 weeks of age. MC38 and CT26 tumors were inoculated into C57BL / 6J and BALB / c mice, respectively. For subcutaneous (s.c.) inoculation, cells were dispersed in Dulbecco’s Modified Eagle Medium (DMEM; 106cells in 100 μL) and injected to the left abdomen of the mice. Orthotopic inoculation was performed by surgery as reported (Uccello, T. P. et al. Development of an Orthotopic Murine Model of Rectal Cancer in Conjunction With Targeted Short-Course Radiation Therapy. Adv. Radiat. Oncol. 7, 100867 (2022); Greenlee, J. D. & King, M. R. A syngeneic MC38 orthotopic mouse model of colorectal cancer metastasis. Biol. Methods Protoc. 7, (2022). Mice were anesthetized under 2.5% isoflurane. Abdominal incision was made to expose the cecum. MC38 cells were dispersed in DMEM (106cells in 50 μL) and injected into the cecal wall from the serosal side.

[0374] Volume of the s.c. tumors were monitored by calliper measurement. The tumor volume was calculated by:

[0375] V is the tumor volume. L and W are the length and width of the tumor, respectively.

[0376] Example 1 : Synthesis of PEG-pLL(CDM) polymer

[0377] Carboxydimethyl-maleic anhydride (CDM) modified polyethylene glycol)-poly(L- Lysine) (PEG-pLL(CDM)) block copolymer (Formula (la)) was synthesized according to the methods of Chen, P et al. Adv. Sci. 2205139 (2023), the contents of which are incorporated herein by reference in their entirety.

[0378] Briefly, polyethylene glycol)-poly(L-Lysine) (PEG-pLL) was synthesized by aqueous N-carboxyanhydride (NCA) ring-opening polymerization. MeO-PEG-NH2(Mw = 12 000; 500 mg) was dissolved in 10 mL 50 x 10-3m NaHCO3solution (pH = 8.4). Lys(TFA)-NCA (500 mg) was added to the ice-bathed MeO-PEG-NH2solution with fast stirring. The mixture was kept reacting in ice bath for 12 h, followed by dialysis (membrane MWCO = 6000-8000) against pure water, and then lyophilized. The lyophilized powder was redissolved in methanol and precipitated using diethyl ether to afford purified poly(ethylene glycol)- poly(trifluoroacetyl-L-Lysine) (PEG-pLL(TFA)). The product was characterized by1H NMR in dimethyl sulfoxide (DMSO)-d6and gel permeation chromatography (GPC; column: TSK gel G4000HHR; mobile phase: DMF; flow rate: 0.75 mL min-1; detector: UV 220 nm) to confirm the successful polymerization. The PEGpLL(TFA) was dissolved in methanol containing 1 N NaOH for 12 h reaction to remove the trifluoroacetic acid (TFA) group. The mixture was purified by dialysis (membrane MWCO = 6000-8000) against pure water. After lyophilization, PEGpLL was obtained as a white solid. The PEG-pLL was characterized by1H NMR in D2O and high-performance liquid chromatography (HPLC; column: Superdex 200- 10 / 300GL; mobile phase: pH 7.4 D-PBS; flow rate: 0.75 mL min-1; detector: UV 220 nm). Next, carboxy dimethylmaleic anhydride (CDM) (100 mg) was dissolved in 8 mL CH2CI2and added with 2 mL oxalyl chloride and allowed to stir for 12 h. The mixture was dried under vacuum to remove excess oxalyl chloride and CH2CI2to afford CDM-C1 as an oil. CDM-C1 was then redissolved in 2 mL CH2CI2and added with 8 mL CH2CI2containing PEG-pLL (100 mg) for a 12 h reaction. The final product, PEGpLL(CDM), was collected by precipitating the mixture using diethyl ether. PEG-pLL(CDM) was characterized by 'H NMR in DMSO-d6and HPLC (column: Superdex 200-10 / 300GL; mobile phase: pH 3.3 acetate buffered saline with 10 x 10-3 m acetate and 500 x 10-3 m NaCl; flow rate: 0.75 mL min-1; detector: UV 220 nm). FIG. 7 is an1H-NMR of the PEG-pLL(CDM) polymer. The length of the pLL block was determined by comparing the integration of the peaks of the -CH2-CH2on the group derived from PEG (δ = 3.5 ppm) and the -CH2-CH2-CH2 of the lysine residue (δ = 1.2-1.8 ppm, peak 2, 3, 4) and calculated to be ~ 40 units. The number of CDM groups on the polymer was calculated by the peak of -CH3 (δ = 2.0 ppm, peak 6) and determined to be 15. FIG. 8 shows a size exclusion chromatography (SEC) of the PEG-pLL(CDM) polymer. The result confirmed narrow molecular size distribution of the polymer.

[0379] Example 2: Preparation of IL-15Rα complex-loaded micelles and Physical characterization of Nano-IL-15 superagonist (Fluorescence cross correlation spectroscopy)

[0380] A functional block co-polymer with a polyethylene glycol)-poly(L-Lysine) (PEG- pLL) backbone was utilized to concurrently associate IL- 15 and IL-15Rα. The modified pLL block of the polymer simultaneously complexes IL- 15 and IL-15Rα, bridging their coordination and further assembles into nanostructure encapsulating the IL-15 / IL-15Rα complex (IL-15cx), namely IL-15 nanosuperagonist (Nano-SA) (FIG. 1A). This Nano-SA stabilizes and protects the IL- 15 / IL-15Rα complex during circulation. Upon systemic administration, Nano-SA displays improved pharmacokinetics and retains intact IL-15cx during circulation. However, Nano-SA can sense the acidic tumor microenvironment due to the pH- sensitivity of the polymer, ensuring a tumor-targeted delivery of IL-15cx. Thus, Nano- SA avoids off-target systemic immune activation but effectively potentiates on-target intratumoral immune activation.

[0381] Nano-IL-15 superagonist was generated by coating complexes of IL- 15 and IL-15Rα (IL-15cx) with a pH-sensitive biocompatible polymer (for preparation of the polymer, see Example 1). The CDM-modified PEG-pLL (PEG-pLL(CDM) block copolymer was concurrently anchored to IL-15cx through pH-sensitive covalent bonds formed between CDM groups and the amines on the surface of the proteins (e.g., the amines of the lysine residues). Moreover, the coating was further stabilized by pH-sensitive crosslinking between the amine moieties and CDM groups in the polymers.

[0382] Preparation and characterization of Nano-SA

[0383] IL-15cx was prepared by mixing IL-15 and IL-15Rα (100 pM for each) in phosphate buffer (10 mM, pH 7.4) and stabilized at 25 °C for 30 min before following operations. To prepare Nano-SA, PEG-pLL(CDM) (10 mg mL-1) was dissolved in acidic phosphate buffer (20 mM, pH 5.0). IL-15cx (40 μg mL-1) was diluted in basic phosphate buffer (20 mM, pH 8.0). The polymer solution was pumped into IL-15cx solution at a rate of 2 μL min-1until the molar ratio of polymer to IL-15cx reached 200:1. The mixture was then titrated to pH 7.4 with phosphate buffer, followed by 6 h incubation at 4 °C. The samples were purified and concentrated by centrifugal filter (100 000 MWCO). SDS-PAGE of the samples were conducted in MES-SDS running buffer under 200 V for 25 min. The gels were stained with 0.1% Coomassie Brilliant Blue R-250 solution to visualize the bands. FIG. IB shows SDS- PAGE of free proteins and Nano-SA. Nano-SA showed significantly reduced mobility in electric field, indicating a giant molecular size.

[0384] Fluorescence correlation spectroscopy (FCS) measurement of the samples was conducted by a laser scattering confocal microscope (CLSM). In the samples for FCS measurement, IL- 15 was labelled with A647 and IL-15Rα was labelled with A488 (see Labelling of Proteins and Polymers General Method). IL-15cx or Nano-SA were diluted in HEPES buffer (10 mM, pH 7.4) to 10 μg mL-1with an equivalent concentration of IL-15cx and loaded to 8-well chambered slides for FCS scanning by 633 and 488 nm excitation lasers alternatively. The autocorrelation function curves of the samples in both observation channels were fit by ZEN software to calculate the diffusion kinetics-related parameters. For TME (JEM-1400, JEOL, Japan), Nano-SA were dispersed in E1EPES buffer (10 mM, pH 7.4) and stained by uranyl acetate, then placed on 400-mesh copper grides for observation. DLS and potential measurement were conducted in zetasizer (Nano-ZS, Malvern, UK) with samples dispersed in HEPES buffer (10 mM, pH 7.4). To analyse the formation of the IL-15cx and the Nano-SA, the diffusion of IL-15 and IL-15Rα was measured by fluorescence correlation spectroscopy (FCS) after labelling IL- 15 and IL-15Rα with AlexaFluor 647 (A647) and AlexaFluor 488 (A488), respectively. The results showed high consistency in the autocorrelation function (ACF) curves and the diffusion time in the two fluorescence channels (FIG. 1C and ID). Moreover, the formation of the IL-15cx in the solution was confirmed by cross-correlation analysis (correlation coefficient C = 1.050; FIGs. 9A-B). The stoichiometric ratio of IL- 15 to IL-15Rα in IL- 15 ex was further validated by comparing the fluorescence intensities from single molecules with the standard samples of IL- 15 or IL-15Rα monomers (Table 2). The FCS results of Nano-SA showed delayed diffusion kinetics in both fluorescence channels, evidenced by the lagged ACF curves and increased diffusion time (FIGs. 1C and ID), which indicate the incorporation of IL-15cx in larger molecular structures. The cross-correlation analysis of Nano-SA also displayed high correlation (C = 1.072,) in the two fluorescence channels (FIGs. 9A-B), demonstrating the strict coencapsulation of one IL- 15 and one IL-15Rα in single Nano-SA.

[0385] The structure of Nano-SA was further characterized by transmission electron microscopy (TEM), in which the Nano-SA appeared to be uniform dark dots after staining with uranyl acetate with a diameter = 21 ± 5 nm (FIGs. IE and IF). As the PEG block of the polymer is hardly visualized under TEM observation due to its low electron density, these black dots represent the core structure of the Nano-SA, consisting of IL-15cx and pLL blocks. Dynamic light scattering (DLS) determined the hydrodynamic diameter of Nano-SA to be 39 ± 3.0 nm (FIGs. 1G and 1H). The difference between the core size and hydrodynamic size indicates a dense PEG layer of around 10 nm on the surface of Nano-SA, which effectively shields the proteins and masks their surface charge (FIG. II). Table 2. FCS characterization of proteins and Nano-SA

[0386] Example 3: Physical characterization ofNano-IL-15 superagonist micelle (Micelle Stability by FRET Analysis In vitro / In vivo)

[0387] In this example, the stability of the Nano-IL-15 superagonist micelle (Nano-SA) was measured by Forster resonance energy transfer (FRET) analysis. The IL-15-Rα complex was labeled on IL-15 with A647 and IL-15-Rα with AlexaFluor 555 (A55) (see Labelling of Proteins and Polymers General Method), thus forming a FRET pair.

[0388] FIG. 2A is a schematic illustration of the FRET-based probe system for tracking IL- 15cx stability. The FRET signal between IL- 15 and IL-15Rα indicates the association state between the two components.

[0389] Stability of IL-15cx or Nano-SA was evaluated in murine plasma. Blood was collected from healthy BALB / c (6 weeks) mice from the abdominal aorta and treated with heparin. Plasma was separated after 2000 x g 15 min centrifuge.

[0390] FCS measurements of samples incubated in plasma were performed. Samples formed by A647-labelled IL-15 and A488-labelled IL-15Rα were diluted in plasma to 10 μg mL-1IL- 15cx equivalent concentration and incubated at 25 °C. After 24 h incubation, samples were measured by FCS.

[0391] To quantify the association of IL- 15 with IL-15Rα, a FRET-based probe system was developed, in which IL- 15 was labelled with A647 and IL-15Rα was labelled with A555. Fluorescence spectra of the samples were measurement by a fluorescence spectrometer with 555 nm excitation. The FRET signal was defined as:

[0392] F refers to the FRET signal. E677and E570are the fluorescence emission intensity of the sample at 670 nm and 570 nm, respectively. The FRET-based probe samples were first tested with a reconstitution experiment, in which they were diluted to 50 μg / mL IL-15cx equivalent concentration in phosphate-buffered saline (PBS; 10 mM phosphate and 150 mM NaCl), and supplemented with equivalent non-labelled IL- 15 or IL-15Rα. After incubation at 37 °C for 2 h, the samples were measured by spectrometer to quantify the FRET signal. To evaluate the stability of the samples in a physiological environment, the samples were diluted in plasma to 50 μg / mL IL-15cx equivalent concentration and incubated under 37 °C . At different incubation times, FRET signals from the samples were measured.

[0393] Nano-SA samples treated by different pH conditions were prepared for pH- sensitivity test. Briefly, Nano-SA (100 μg / mL IL-15cx equivalent concentration) was dissolved in phosphate buffered saline (PBS) (10 mM phosphate and 500mM NaCl) with different pH and incubated at 4 °C for 24 h. SDS-PAGE and FCS measurement of the samples were conducted. In FCS analysis, the sample diffusion time observed for the 633 nm excitation channel was used for calculating the diffusion coefficient and estimating the hydrodynamic diameter of the sample via Einstein- Stokes equitation, as shown in the following formulas:

[0394] Dsampleand tsamplerefer to the diffusion coefficient and measured diffusion time of the sample, respectively. DA647and tA647are the diffusion coefficient and measured diffusion time of free A647 dye standard, in which DA647= 3.05 x 10-6cm2s-1according to reference (Derouard, J., Wang, I., Delon, A. & Leroux, C.-E. Adaptive optics for fluorescence correlation spectroscopy. Opt. Express, Vol. 19, Issue 27, pp. 26839-26849 19, 26839-26849 (2011)). D is the hydrodynamic diameter of the sample. KBis Boltzmann constant. T is the temperature during experiment. η is the viscosity of the buffer.

[0395] For estimating the activation fraction of Nano-SA, standard samples were prepared and measured by FCS. Freshly prepared Nano-SA was measured to represent the standard result of intact particles (remaining micelles = 100%). Free IL-15cx was measured to represent the standard result of completely activated particles (remaining micelles = 0%). The hydrodynamic diameter was retrieved from FCS measurement. The remaining micelle fraction was calculated from the following formula: dsampleis the hydrodynamic diameter of the sample. d100%and d0%are the hydrodynamic diameters of intact Nano-SA and free IL-15cx, respectively.

[0396] The release profile of Nano-SA was evaluated by dialysis. Nano-SA loading A647- labelled IT-15 and A488-labelled IL-15Rα were dissolved in pH 7.4 PBS (10 mM phosphate and 500mM NaCl) and loaded into dialysis cassettes (MWCO= 20 000 and 100 000). The cassettes were stored in 1 L PBS (10 mM phosphate and 500mM NaCl, pH = 7.4 or 6.5) and incubated at 25 °C . Samples were then collected from the cassettes. The fluorescence intensity from A647 was measured with a fluorescence spectrometer to indicate the remaining amount of IL- 15 in the system.

[0397] Enzyme-linked immunosorbent assay (ELISA) and cell experiments were conducted to evaluate the binding affinity of Nano-SA to targets. Activated Nano-SA sample was prepared by dissolving Nano-SA (100 μg / mL IL-15cx equivalent concentration) in acidic PBS (10 mM phosphate and 500 mM NaCl, pH 6.5) and incubating at 4 °C for 24 h. In ELISA, samples were parallelly diluted to gradient concentrations and analyzed by the protocol provided by the manufacturer. In the cell experiment, KHYG-1 cells (106cells / mL) were incubated with fluorescence-labelled samples (10 μg / mL) in PBS (pH 7.4, 10 mM phosphate and 150 mM NaCl) containing 2% fetal bovine serum (FBS) at 4 °C for 1 h. After washing with PBS with centrifugation at 500 xg for 5 min, cells were dispersed in 10 μg / mL Hoechst 33342 PBS solution and imaged by CLSM (excitation: 405, 488, 633 nm, detection: 460, 510, 670 nm respectively). Cells were also analyzed by a flow cytometer equipped with fluorescein isothiocyanate (FITC) and Cy5 filter sets to quantify the fluorescence intensities. Bioactivity was evaluated with KHYG-1 cells. KHYG-1 cells were seeded into 96-well plates (104cells per well) and samples were added into the medium at varying concentrations. After 6 h of incubation, the plate was centrifuged (500 g x5 min) and replaced with fresh medium. After 24 h more of incubation, the cell viability was determined by cell counting kit (CCK)-8.

[0398] FIGs. 10A-C show a reconstitution test of FRET-based formulations confirms the protection of Nano-SA on the association between IL- 15 and IL- 15Rα. FIG. 10A is a schematic illustration of the experiment design. Free IL-15cx or Nano-SA was prepared with A647- labelled IL- 15 and A555-labelled IL- 15Rα. The samples were incubated with non-labelled IL- 15 or IL-15Rα for reconstitution. The FRET signal loss could reflect the reconstitution degree. FIG. 10B shows fluorescence emission spectra of the samples with excitation light at 555 nm. Intensities were normalized to the emission at 570 nm. FIG. 10C shows FRET signals of the samples. Data are plotted as the mean ± S.D., n = 3 independent measurements.

[0399] In murine plasma, a significant decrease in the FRET signal was observed for IL- 15cx, indicating its instability under physiological conditions (FIG. 2B). In contrast, Nano- SA maintained a high FRET signal, confirming its capability to protect IL-15cx. Additionally, FCS-based cross-correlation analysis (FIG. 11) showed that Nano-SA maintained high cross-correlation level similar to the initial state, while IL-15cx exhibited a clear loss of cross-correlation.

[0400] To assess stability of Nano-SA at different pH levels, SDS-PAGE analysis of Nano- SA incubated under different pH conditions was performed. SDS-PAGE analysis showed cargo release from micelles at pH 6.5 and 7.0 (which can correspond to the intratumoral environment), but not at pH 7.4, which approximates the homeostatic circulatory pH (FIG. 2C). Moreover, the changes in the hydrodynamic size of Nano-SA at different pHs was analyzed by FCS. The results showed a sudden decrease of the size was observed at pHs lower than 7.0 (FIG. 2D), along with a reduction in the overall percentage of intact micelles (FIG. 2E). In addition, the release profile of IL-15cx from Nano-SA was studied by dialysis at pH 7.4 and 6.5 (FIG. 2F). Protein release was observed at pH 6.5, but not at 7.4. Moreover, the released proteins were unable to pass through the dialysis membrane with a molecular weight cut-off of 20 kDa, indicating that the released species existed as IL-15cx rather than IL- 15 monomers.

[0401] ELISA confirmed that Nano-SA efficiently masked IL-15cx from interacting with detection molecules (FIG. 2G). However, upon activation in acidic buffer, Nano-SA restored its affinity to the same level as native IL-15cx. In KHYG-1 cells, the intact Nano-SA prevented the binding of IL-15cx to the surface of cells, while activated Nano-SA regained the binding affinity to the cells (FIGs. 2H and 21). Thus, Nano-SA promoted a switchable agonistic function in the proliferation of KHYG-1 cells (FIG. 2J). Comparing to monomeric IL-15, IL-15cx and activated Nano-SA showed a stronger stimulative effect on KHYG-1 cell proliferation, confirming the superagonistic potency.

[0402] Example 4: Pharmacokinetics and Biodistribution of Nano-IL-15 superagonist micelles

[0403] Blood circulation profiles of IL-15cx and Nano-SA were determined by intravital confocal microscopy (IVCLSM) in healthy C57BL / 6J mice (6 weeks age). A647-labelled IL- 15 and A488-labelled IL-15Rα (see Labelling of Proteins and Polymers General Method) were used to prepare the samples. Samples containing 10 μg IL-15cx equivalence were dispersed in 100 μL pH 7.4 PBS (10 mM phosphate and 150 mM NaCl) and injected into mice through the tail vein. The earlobe skin of the mice was then observed by IVCLSM to monitor the fluorescence signal (excitation: 488 and 633 nm, detection: 510 and 670 nm respectively). For quantification, regions of interest (ROIs) were placed in vessels, and the mean fluorescence intensities in the ROIs were plotted against time. Definite intensity values were normalized by the maximum intensity found in each ROI. A biodistribution study was performed in C57BL / 6J mice bearing s.c. MC38 tumors. Mice were injected with the same samples from the former IVCLSM experiment. Eight hours post injection, mice were sacrificed to harvest the tissue samples, including organs and tumors. Samples were imaged by in vivo imaging system (IVIS). Quantified biodistribution profiles were measured by tissue homogenization. Tissue samples were homogenized by multibeads homogenizer in protein extraction buffer. Supernatants were collected after centrifugation (10,000 x g for 10 min). Fluorescence intensity in the sample was measured by a fluorescence spectrometer. IL- 15 and IL-15Rα solutions were used as standards for calculating the concentrations of the samples.

[0404] After injection, Nano-SA remained in blood circulation and avoided extravasation in the skin (FIGs. 3A and 3B). The quantification of the blood circulation profile showed that Nano-SA extended the fluorescence intensities of both A647-labelled IL- 15 and A488- labelled IL-15Rα in the vessels for more than 2 h (FIG. 3C). In contrast, IL-15cx was rapidly cleared and extravasated from the vessels into the skin tissue (FIGs. 3A, 3B and 3C).

[0405] In vivo FRET

[0406] The in vivo stability of IL- 15 ex was evaluated by the FRET -based probe system by IVCLSM with a method (method was similar to method described in Watanabe, S. et al. In vivo rendezvous of small nucleic acid drugs with charge-matched block catiomers to target cancers. Nat. Commun. 2019 101 10, 1 13 (2019)). Samples containing 10 μg IL-15cx equivalence were dispersed in 100 μL pH 7.4 PBS (10 mM phosphate and 150 mM NaCl) and injected into healthy C57BL / 6J mice through the tail vein. The earlobe skin of the mice was observed by IVCLSM using a FRET observation mode to record spectral images (excitation: 555 nm; detection: channel I: 570-600 nm, channel II: 650-700 nm). The results were analyzed by NIS Elements Software. The spectral images were linearly unmixed to separate the two channels, and the FRET signal was defined as:

[0407] F is FRET signal. A UC\ and A UCF are the area under the curve of channel I spectrum and channel II spectrum, respectively. ROIs were placed in vessels for plotting the mean FRET signal against time.

[0408] After intravenous (i.v.) injection of the IL-15cx FRET pair, only a transient FRET signal was observed in the vessel, which vanished after 10 min (FIGs. 3D, 3E, and 13). On the other hand, Nano-SA displayed stable FRET signal in the vessels that lasted for over 200 min. These results indicate that Nano-SA maintained the IL-15 complex during circulation in blood, whereas IL-15cx is immediately disrupted in the bloodstream.

[0409] The biodistribution of Nano-SA in mice bearing MC38 tumors after i.v. injection was also evaluated. Nano-SA promoted threefold higher fluorescence intensity in tumors from both A647 and A488 channels than IL-15cx (FIG. 3E). In the organs, Nano-SA showed comparable distribution of IL- 15 and IL-15Rα (FIGs. 3F, 3G, and 3H). Distribution of the components of IL- 15cx was different in the lungs, which showed higher IL- 15 levels than IL- 15Rα levels, and in the kidneys, which presented higher IL-15Rα levels than IL- 15 levels (FIGs. 3G and 3H). Such discrepancy in the biodistributions of IL-15 and IL-15Rα in different organs further supports the dissociation of the IL- 15 ex.

[0410] Example 5: Demonstration of pH activated release of IL- 15 -Rα complex in vivo

[0411] The tumor micro environment is characterized, inter alia, by lower pH values than normal tissue. The use of acid-labile functional groups is one possible mechanism to increase the targeting of therapeutic agents to a tumor.

[0412] In this example, the stimuli-responsive (e.g., acid-labile) properties of the Nano-IL-15 superagonist particles were demonstrated. The Nano-IL-15 superagonist (Nano-SA) particles were prepared as described in Example 2 with the addition of QSY-21 to the block copolymer. QSY-21 is a non-fluorescent molecule with a broad and intense absorption band that peaks at 661 nm. The resulting Nano-IL-15 superagonist particles will exhibit fluorescence quenching with respect to the A647 fluorophore.

[0413] An activation indicator formulation of Nano-SA, namely Al -Nano-SA was prepared. QSY-21 -labelled polymer, A647-labelled IL-15, and A488-labelled IL-15Rα (see FIG. 4A and Labelling of Proteins and Polymers General Method) were used to prepare the formulation with the same protocol of preparing Nano-SA. The dissociation-dependent fluorescence of Nano-SA was confirmed in vitro. AI-Nano-SA (10 μg mL-1) were dispersed in PBS (10 mM phosphate and 500mM NaCl) with different pH (6.5 and 7.4). The fluorescence intensities at 510 and 670 nm were measured by a fluorescence spectrometer.

[0414] For the in vivo study, AI-Nano-SA containing 10 μg IL-15cx equivalence were dispersed in 100 μLpH 7.4 PBS (10 mM phosphate and 150mM NaCl) and injected into mice bearing CT26 or MC38 tumors via the tail vein. Mice were observed by IVIS (filter: Ex640 / Em680) at different time points. After 12 h, the mice were scarified. Tissue samples were excised for ex vivo imaging (filters: Ex480 / Em510 and Ex640 / Em660). Fluorescence intensity from each tissue sample was quantified from the images. For a dynamic observation from sub-tissue level, IVCLSM (excitation: 488 and 633 nm, detection: 510 and 670 nm respectively) was used in mice bearing MC38 tumors. Earlobe skin and tumor tissue were observed by IVCLSM. In the case of tumor observation, the skin covering the tumor area was removed. The images from the red channel and the green channel were analyzed by Image J software to calculate the intensity ratio of red to green fluorescence (R / G). Six hours after injection, the earlobe skin or tumor tissues were imaged from different layers for a Z-stacking sampling to reconstitute a 3D mapping of the tissues with totally 100 pm depth.

[0415] To visualize the polymer stripping from Nano-SA in situ, an activation indicator of Nano-SA (AI-Nano-SA) that can be traced in in vivo settings was produced (FIG. 4A). AI- Nano-SA was achieved by introducing a quencher of A647 (QSY-21) in the PEG-pLL(CDM) polymer (FIG. 12A) and engaging with the IL-15cx made from A647-labelled IL-15 and A488-labelled IL-15Rα. In this formulation, the fluorescence from A647 (red fluorescence) is transiently quenched by QSY-21 at physiological conditions, while the fluorescence from A488 (green fluorescence) is always visible. Upon polymer detachment at low pH, AI-Nano- SA is de-quenched, and the red fluorescence is retrieved (FIG. 12B). Thus, the intensity ratio of red to green fluorescence can be used to indicate the activation level of Nano-SA. AI- Nano-SA was intravenously injected into tumor-bearing mice. The red fluorescence signal was observed from the tumor area by IVIS, and the signal intensity showed a time-dependent escalation, indicating the gradual activation of AI-Nano-SA in tumors (FIG. 4B). Twelve- hours post injection, the tissues were collected and imaged ex vivo by IVIS. The distribution of green fluorescence indicates the distribution of AI-Nano-SA, and the red fluorescence reflects the activation of AI-Nano-SA in the tissues (FIG. 4C). Quantitative assessment of the red to green fluorescence (R / G) ratio showed significantly higher activation of the AI-Nano- SA in tumors than in other tissues (FIG. 4D), confirming the tumor selectivity of the formulation. These results indicate AI-Nano-SA is stable during blood circulation and in healthy tissues. In the tumor, the green fluorescence of AI-Nano-SA was evident within both the blood vessels after intravenous injection, indicating that the formulation reaches the tumor in intact form. As time passed, AI-Nano-SA extravasated into the tumor tissue, where the acidic intratumoral pH stripped the polymer coating, retrieving the red fluorescence (FIG. 4E). Both the green and red signals increased with time, as more AI-Nano-SA accessed the tumor and got activated. Quantitative analysis of the R / G ratio confirmed the activation degree of AI-Nano-SA augmented with time. A three-dimensional reconstruction from Z- stack images of the observation area also showed thorough distribution of activated AI-Nano- SA in the tumor tissue (FIG. 4F). The results confirmed the selective release of IL-15cx into the tumor microenvironment.

[0416] Example 6: Demonstration ofNano-IL-15 superagonist tumor-targeted immunomodulation

[0417] The tumor specificity of the immune responses triggered by Nano-IL-15 superagonist (Nano-SA) and IL-15cx were evaluated in mice bearing s.c. MC38 or CT26 tumors. IL-15cx or Nano-SA treatment was conducted in different dosing schedules as described in FIG. 5A. Blood samples were collected from the abdominal aortas and treated by heparin. For ELISA analysis, blood samples were centrifuged at 2000 x g for 15 min to collect the plasma and measured by ELISA kits using the manufacturer’s protocol. Blood, spleen, liver, and tumor samples were analyzed by flow cytometry. Spleen samples were mechanically smashed to prepare cell suspensions and red blood cells were removed using RBC lysis buffer using manufacturer’s protocol. Liver samples were digested using collagenase IV and the leukocytes were enriched by density-differentiation centrifuge with Percoll via reported method (Shi, W. et al. Isolation and purification of immune cells from the liver. Int. Immunopharmacol. 85, (2020)). Tumor samples were digested using collagenase IV To stain for membrane-associated markers, cells were incubated with fluorescence-conjugated antibodies in PBS (pH 7.4, 10 mM phosphate and 150 mM NaCl) containing 2% FBS under 37°C for 1 h. For staining intracellular markers, cells were fixed and permeabilized using a Foxp3 staining buffer set, and stained with antibodies against the intracellular markers. Gating strategies of the flow cytometry analyses were summarized in FIGs. 14 and 16. NK cells in liver and spleen samples were sorted and used for in vitro killing assay against YAC-1 cells based on the method reported in Sugimoto, C. et al. A flow-cytometry-based assay to assess the cytolytic activity against tumor cells by combination of mouse MAIT cells and natural killer cells. STAR Protoc. 4, (2023). YAC-1 cells were stained with PKH26 then cocultured with lymphocytes. For splenocytes samples, the ratio between splenocytes and YAC- 1 cell number (effector / target cell ratio) was 65:1. For liver leukocyte samples, the effector / target cell ratio was 6:1. After 4 h co-incubation, the cells were stained with Annexin- V and 7-AAD. The samples were analyzed by flow cytometer.

[0418] For histological analyses, fresh tumors excised from the mice were frozen in optimal cutting temperature (O.C.T.) compound and sliced by a Cryostat to 10 pm thickness. Hematoxylin and eosin (H&E) staining was performed with the protocol provided by manufacturer of the reagents and imaged by a microscope. Immunofluorescence (IF) staining of NKp46, CD8α, PD-L1 and granzyme B was performed on tumor sections. For staining, NKp46, CD8α and PD-L1 corresponding fluorescence-conjugated antibodies were diluted in PBS (pH 7.4, 10 mM phosphate and 150 mM NaCl) containing 2% FBS with the concentration suggested by the manufacturer. For staining granzyme B, the sections were fixed and permeabilized by Foxp3 staining buffer set and stained with FITC-conjugated granzyme B antibody with the protocol suggested by the manufacturer. IF-stained sections were mounted in antifade mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) and imaged by CLSM (excitation: 405, 488, 633 nm, detection: 460, 510, 670 nm respectively).

[0419] For measuring the intratumoral cytokines, tumor samples were homogenized using a multibead homogenizer in protein extraction buffer supplemented with a protease inhibitor cocktail. Supernatants were collected after centrifugation at 10000 x g for 10 min. Cytokine concentrations in the supernatants were measured by ELISA kits.

[0420] After injection of Nano-SA or IL-15cx (3 i.v. injections; 10 μg IL-15cx equivalence (e.q.) per injection), blood, liver and spleen were collected from mice for immune analysis (FIG. 5A). In mice treated with IL-15cx, downstream cytokines like interferon-γ (IFN-γ) and interleukin-6 (IL-6) were upregulated in blood (FIG. 5B). Additionally, antagonists of IL- 15, such as interleukin- 10 (IL-10) and transforming growth factor-P (TGF-P) were also upregulated. Conversely, Nano-SA significantly reduced the downstream cytokine responses in blood (FIG. 5B), demonstrating a lower systemic immune response.

[0421] The immune cell subtypes in blood were also screened to assess immune activation. Counts and activation of NK cells, which are the primary IL- 15 -responsive immune cells ,were assessed. IL-15cx significantly expanded NK cell number in blood circulation, whereas the level of NK cells for Nano-SA remained close to PBS-treated mice (FIG. 5C). Moreover, IL-15cx upregulated the CD69 expression in NK cells (FIG. 5D), indicating that the cells were activated. Similar results were observed in spleen (FIG. 5E) and splenomegaly induced by IL-15cx was also observed (FIG. 5F), indicating IL-15-associated immunotoxicity. Increased NK cell frequency was also found in the liver of mice treated with IL-15cx, but not for Nano-SA (FIG. 5G).

[0422] To further evaluate the activity of the NK cells in spleen and liver, the in vitro cytotoxicity against YAC-1 model cells was tested (FIGs. 14 and 15). Briefly, NK cells were sorted from the spleen and liver samples by flow-cytometer, and co-incubated with YAC-1 cells, a murine lymphoma cell line which is sensitive to be killed by NK cells. The NK cells collected from the spleen and liver of mice treated with IL-15cx killed the YAC-1 cells (FIG. 14), inferring the potential risk for inducing organ damage. On the other hand, the cells from Nano-SA-treated mice showed lower cytotoxicity against YAC-1 cells.

[0423] Another significantly altered cell type by IL- 15 ex treatment were T cells. In blood, spleen and liver, both CD4+ and CD8+ T cells showed significantly upregulated CD69 and Ki67 expression (FIGs. 15-19). Nano-SA treatment induced a much lower response. Together, these results indicate that Nano-SA prevented systemic immune response induced by IL-15cx (FIG. 20).

[0424] In tumors, the effects of IL-15cx and Nano-SA were opposite to those observed systemically. The infiltration of immune cells in tumors after IL- 15cx was similar to the control group (FIGs. 5H-5J). Nano-SA led to the expansion of elfector cells, including NK, NKT and CD8+ T cells in the tumor microenvironment. Similar results were observed in mice bearing CT26 tumors (FIGs. 21-24), in which IL-15cx induced a stronger systemic response but lower intratumoral immune infiltration compared to Nano-SA.

[0425] The cytokine response in CT26 tumor tissue was also evaluated (FIG. 23). IFN-γ and IL-6 were enriched in tumor by Nano-SA treatment, indicating an enhanced intratumoral inflammation response. IL-15cx induced higher levels of IL-10 and TGL-β These findings indicate that Nano-SA enhanced the immune agonism in tumor, achieving a tumor-targeted immunomodulation.

[0426] Table 3. Summary of the antibodies and dyes used for flow cytometry experiments

[0427] Example 7: Demonstration ofNano-IL-15 superagonist antitumoral efficacy, safety evaluation, and synergy with immune checkpoint inhibitors

[0428] Antitumoral efficacy and safety profile of Nano-SA were evaluated in different animal models and different treatments. Details of the experiment schedule are depicted in FIG. 6 A. In combination therapies, anti-PD-L1 antibodies were dispersed in saline (100 μg in 100 μL) and injected intraperitoneally (i.p.). Subcutaneous tumor volumes were measured with a caliper. Mice were euthanized after the tumor volume exceeded 1000 mm3and counted as death in the survival curves. Bodyweight of the mice were recorded during the experiments.

[0429] Blood and tissue samples were collected at a determined time during the treatment. Blood was collected from orbital vein by capillary tube and left under room temperature for 20 min to coagulate, then centrifuged (10000 x g 10 min) to separate the serum. IFN-γ in serum was measured by ELISA. Organ damage-associated biomarkers in serum were analysed with a blood chemistry analyzer. Tissue samples were sectioned for H&E staining. For tumor sections, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay staining was also performed using the manufacturer’s protocol.

[0430] Histological analysis of the tumor sections after Nano-SA treatment showed tissue damage in H&E staining, and the TUNEL assay confirmed the stronger apoptotic / necrotic process of tumor cells by Nano-SA treatment (FIG. 6B). Thus, Nano-SA exhibited more effective inhibition of the tumor growth than IL- 15 ex, resulting in prolonged survival of the animals and complete response (CR) in 4 out of 5 mice in the group (FIGs. 6C and 6D). IL- 15cx performed similarly to the control group. Moreover, during the IL-15cx treatment, the bodyweight of the animals dropped immediately after the first injection (FIGs. 25A-C), indicating IL- 15 ex toxicity.

[0431] Serum IFN-γ was measured as an indicator of systemic immune activation before and after the treatments. IL- 15 ex induced an average 7.5-fold increase in serum IFN-γ over the baseline. The response from Nano-SA was not significantly different from the control group (FIG. 26). Moreover, histological analysis and serum biomarkers measurements were performed to assess the organ damage level. In H&E staining, no clear pathological changes were identified in heart, liver, lung and kidney for both IL-15cx and Nano-SA (FIG. 27). However, the morphology of the white pulp in spleen of the mice treated with IL- 15 ex was different from the control and the Nano-SA-treated mice. The boundaries between red and white pulps of the spleens in the IL-15cx group were not clear, indicating immunotoxicity from IL-15cx. In addition, the level of serum biomarkers of liver damage, including total protein (TP) and alanine aminotransferase (ALT), showed a significant increase in IL- 15 ex treated mice, but not for mice receiving Nano-SA (FIG. 28). The results demonstrated the capability of Nano-SA to achieve effective and safe immunotherapy in MC38 tumors.

[0432] The performance of Nano-SA was further studied in an orthotopic (o.t.) MC38 colon tumor model (FIG. 6E). Nano-SA was able to significantly inhibit the tumor progression (FIG. 29), leading to a CR in 3 out of 5 mice. Moreover, therapeutic experiments in CT26 colon tumor model with a lower dose further confirmed the superior efficacy of Nano-SA over IL-15cx (FIGs. 30A-30B). Synergy with Immune Checkpoint Molecules

[0433] Nano-SA upregulated PD-L1 in the tumor microenvironment (FIG. 6F). Thus Nano- SA was combined with anti-PD-L1 antibody (aPD-L1) to treat MC38 tumors (FIG. 6G). To compare the combination and monotherapies, a lower dose (3 μg IL- 15 e.q. per injection) was used. In this dose, IL-15cx, either as a monotherapy or in combination with aPD-L1, performed similarly (FIGs. 6H). However, Nano-SA monotherapy induced complete response in 3 out of 6 mice (FIG. 61). Combining aPD-L1 with Nano-SA further improved the activity of CD8+ T cells, as suggested by the higher granzyme B (GB) level (FIG. 6J), promoting a higher complete response rate (5 out of 6 mice). Notably, no significant difference of bodyweight change was noticed, confirming the safety of the treatments (FIGs. 31A-B).The surviving mice also showed a strong immunological memory, as none showed relapsed tumors after rechallenge with a re-inoculation of MC38 cells (FIG. 6K).

Claims

CLAIMS1. A polymeric complex comprising:(i) an immunocomplex comprising:(a) a cytokine; and(b) a soluble cytokine -binding protein; and,(ii) a block copolymer represented by Formula (I):wherein:R1and R2are each independently hydrogen, optionally substituted C1-12alkyl, azide, amine, maleimide, a ligand or a labeling agent;R3is a group derived from a compound represented by Formula (II):wherein Raand Rbare each independently selected from the group consisting of: hydrogen, optionally substituted alkyl, alkenyl, cycloalkyl group, aryl group, aralkyl group, acyl group, heterocyclyl, heterocyclic alkyl, hydroxy, alkoxy, and aryloxy group, orRaand Rbare taken together with the carbon atoms to which they are attached to form an aromatic ring or a cycloalkyl ring, wherein the bond between the carbon atoms to which Raand Rbare attached may be a single bond or a double bond, wherein R3is covalently attached to the adjacent CH2;L1is selected from the group consisting of: -NH-, -C(O)-, -(CH2)p1-NH-, -L2a-(CH2)q1- L3a-, wherein: pl represents an integer of 1 to 6;L2ais selected from the group consisting of: -OC(O)-, -OC(O)NH-, -NHC(O)-, -NHC(O)O-, -NHC(O)NH-, -C(O)NH-, and -C(O)O-;L3ais NH or CO, and ql is an integer of 1 to 6, ml and m2 are each independently an integer of 0 to 500, wherein the sum of ml and m2 is an integer of 10 to 500, m3, m4 and m5 are each independently an integer of 1 to 5 n is an integer of 0 to 500, and the symbol " / " indicates that (m1+m2) units of the respective monomer units shown on the left and right sides of this symbol may be in any sequence.

2. The complex according to claim 1, wherein the compound represented by Formula (II) is at least one of compounds represented by Formulae (Ila) to (Ilg):

3. The complex according to claim 2, wherein the compound represented by formula (II) is a compound represented by Formula (Ila) or (lIb) :

4. The complex according to any one of claims 1-3, wherein R3is selected from the group consisting of:whereindenotes the point of attachment5. The complex according to any one of claims 1-4, wherein R3is:whereindenotes the point of attachment.

6. The complex according to any one of claims 1-5, wherein R1is C1-12alkyl.

7. The complex according to any one of claims 1-6, wherein R2is hydrogen.

8. The complex according to any one of claims 1-7, wherein L1is -(CH2)p1-NH-.

9. The complex according to any one of claims 1-8, wherein m3 is 4.

10. The complex according to any one of claims 1-9, wherein m4 is 4.

11. The complex according to any one of claims 1-10, wherein m5 is 2.

12. The complex according to any one of claims 1-11, wherein R3represents a direct bond to a compound represented by Formula (II).

13. The complex according to any one of claims 1-12, wherein the block copolymer represented by Formula (I) is a block copolymer represented by Formula (la):

14. The complex according to any one of claims 1-13, wherein ml and m2 are each independently an integer of 1 to 500, wherein the sum of ml and m2 is an integer of 10 to 500.

15. The complex according to any one of claims 1-14, wherein n is an integer of 1 to 500.

16. The complex according to any one of claims 1-15, wherein the immunocomplex is covalently bonded to the block copolymer represented by Formula (I).

17. The complex according to claim 16, wherein the covalent bond is cleaved in a pH- dependent manner.

18. The complex according to any one of claims 1-17, wherein the cytokine is a pro- inflammatory cytokine.

19. The complex according to any one of claims 1-18, wherein the soluble cytokinebinding protein is a soluble cytokine receptor or cytokine-binding fragment thereof.

20. The complex according to claim 19, wherein the cytokine and cytokine receptor or fragment thereof are selected from the group consisting of IL- 15 and IL-15Rα, IL-2 and IL- 2R, and IL-4 and IL-4Rα.

21. The complex according to claim 20, wherein the cytokine is an IL- 15 or a receptorbinding fragment thereof and the cytokine receptor or fragment thereof is an IL-15Rα or an IL- 15 -binding fragment thereof.

22. The complex of claim 21, wherein the IL-15Rα or fragment thereof comprises an IL- 15Rα sushi domain.

23. The complex of claim 21 or 22, wherein the IL-15Rα or fragment thereof comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 244-253, and wherein the IL-15Rα or fragment thereof does not comprise a signal peptide.

24. The complex of any one of claims 21-23, wherein the IL- 15 or fragment thereof comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence of SEQ ID NOs: 257 .

25. The complex of any one of claims 1-18, wherein the soluble cytokine-binding protein comprises an antibody or antigen-binding fragment thereof.

26. The complex of claim 25, wherein the cytokine is an IL-7 and the antibody or antigenbinding fragment is an anti-IL-7 antibody or antigen-binding fragment.

27. The complex of claim 26, wherein the anti-IL-7 antibody or antigen-binding fragment is an M25 antibody or IL-7-binding fragment thereof.

28. The complex of claim 26 or 27, wherein the IL-7 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, and wherein the IL- 15Rα or fragment thereof does not comprise a signal peptide.

29. A pharmaceutical composition comprising the polymeric complex of any one of claims 1-28 and a pharmaceutically acceptable excipient.

30. A method of treating cancer in a subject in need thereof, the method comprising administering the complex of any one of claims 1-28 or the pharmaceutical composition of claim 29 to the subject.

31. The method of claim 30, wherein the cancer is selected from the group consisting of: colorectal cancer, melanoma, colon cancer, breast cancer, lung cancer, pancreatic cancer, brain cancer, head and neck cancer, liver cancer, stomach cancer, and mesothelioma.

32. The method of claim 30 or 31, further comprising administering a checkpoint inhibitor to the subject.

33. The method of claim 32, wherein the checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof.

34. The method of claim 32 or 33, wherein the checkpoint inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

35. The method of any one of claims 30-34, wherein the administration of the complex results in reduced or substantially no off-target toxicity in the subject compared to a reference standard.

36. The method of claim 35, wherein the off-target toxicity is measured by serum cytokine levels; serum ALT, TP, BUN, or CRE levels; and or body weight of the subject37. The method of any one of claims 30-36, wherein the administration of the complex results in reduced or substantially no increase in splenic weight in the subject compared to a reference standard.

38. The method of any one of claims 30-37, wherein the complex exhibits reduced or substantially no disassembly of the immunocomplex compared to a reference standard.

39. The method of any one of claims 35-38, wherein the reference standard comprises the immunocomplex without the block copolymer.

40. A method of delivering an immunocomplex to a cell, the method comprising contacting the cell with the polymeric complex of any one of claims 1-28, wherein a pH level of a region surrounding the cell permits release of cytokine and a cytokine receptor or cytokine-binding fragment thereof.

41. The method of claim 40, wherein the pH level of the region surrounding the cell is about 7.0 or lower42. A method of modulating an immune response in a subject receiving an immunotherapy regimen, the method comprising:(i) administering the complex of any one of claims 1-28 to the subject;(ii) measuring or having measured a biological property of the subject;(iii) changing the course of the immunotherapy regimen based on the measurement of the biological property; thereby modulating the immune response in the subject.

43. The method of claim 42, wherein the complex further comprises a detectable agent.

44. A method of manufacturing the polymeric complex of any one of claims 1-28, the method comprising:(a) contacting the cytokine and the soluble cytokine -binding protein, thereby forming the immunocomplex;(b) contacting (i) a hydrophilic polymer comprising a reactive group and (ii) a plurality of cyclic monomers comprising a basic group under reaction conditions sufficient for a ring-opening polymerization reaction, thereby forming a product of the ring-opening polymerization reaction;(c) contacting the product of the ring-opening polymerization reaction with a charge regulator, thereby forming a block copolymer; and(d) contacting the block copolymer with the immunocomplex, thereby forming the polymeric complex.

45. The method of claim 44, wherein the charge regulator is a compound of Formula (II).

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