Recombinant il-33 proteins and methods of use thereof

Abstract

The present invention provides novel recombinant IL-33 proteins and methods of use thereof. In some embodiments the recombinant IL-33 proteins are Fc fusion proteins comprising an IL-33 domain and an Fc domain.

Description

[0001] Atorney Docket No. MSKCC.064.WO1

[0002] Electronically filed on: January 9, 2026 RECOMBINANT IL-33 PROTEINS AND METHODS OF USE THEREOF

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of priority of U.S. Provisional Patent Application No.

[0005] 63 / 743,620 filed on January 9, 2025, the content of which is hereby incorporated by reference in its entirety.

[0006] SEQUENCE LISTING

[0007] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 8, 2026, is named MSKCC_064_WOl_SL.xml, and is 84,969 bytes in size.

[0008] STATEMENT OF GOVERNMENT SUPPORT

[0009] This invention was made with government support under grant numbers CA262516 and CA257881 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0010] INCORPORATION BY REFERENCE

[0011] For the purposes of only those jurisdictions that permit incorporation by reference, the content of all documents cited herein is hereby incorporated by reference in its entirety. Numbers in parentheses or in superscript following text in this patent disclosure refer to the numbered references provided in the “Reference List” section of this patent specification. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention.

[0012] BACKGROUND OF THE INVENTION

[0013] Interleukin 33 (IL-33) is an IL-1 family alarmin cytokine. While the role of IL-33 in inflammation is well known, it has more recently been discovered that IL-33 may play a role in anti-cancer immunity, in part through activation of immune cells such as DCs [9], cytotoxic anticancer effector T cells [10, 11], NK cells

[0012] , eosinophils [13, 14] and, importantly, group 2 innate lymphoid cells (ILC2s)

[0015] , Wild-type IL-33 may play a role in driving anti-tumorAtorney Docket No. MSKCC.064.WO1

[0014] Electronically filed on: January 9, 2026 immunity either alone or in combination with checkpoint blockade agents

[0015] , However, the wild-type form of IL-33 has limited efficacy as a therapeutic agent because it is rapidly inactivated in the extracellular environment due to oxidation of four unpaired cysteines, neutralization by decoy receptors such as soluble ST2 and IL-lRAcP [11, 12], and proteolytic processing. Accordingly, there is a need in the art for modified forms of IL-33 that overcome these significant obstacles and can be used as therapeutic agents. The present invention addresses these needs.

[0015] SUMMARY OF THE INVENTION

[0016] The present invention is based on a series of important developments and discoveries relating to recombinant IL-33 proteins, further details of which are provided in the Examples, Figures and Detailed Description sections of this patent disclosure. In brief, these developments and discoveries are the result of extensive efforts that were undertaken to rationally design and test novel engineered forms of the mature human IL-33 protein in which: (a) the structure was stabilized using disulfide bonds introduced at sites selected based on crystal structure analysis, (b) sites susceptible to unpaired cysteine oxidation were removed, (c) glycosylation sites were removed, and (d) fusion to a mutated IgG Fc domain was employed. The resulting recombinant IL-33 proteins exhibit enhanced stability, a longer half-life, resistance to cysteine oxidation and proteolysis, and native-like activation of the ST2 / IL-lRAcP pathway, making the novel recombinant IL-33 proteins described herein superior to wild-type IL-33 and other engineered forms of IL-33 for use in various applications. Furthermore, and importantly, the novel recombinant IL-33 proteins described herein also induce ILC2 cell expansion and activation, enhance formation of intra-tumoral tertiary lymphoid structures (TLSs), and effectively control tumor growth control in a preclinical pancreatic cancer model, making the recombinant IL-33 proteins described herein potentially useful as anti-cancer therapeutic agents for the treatment of pancreatic cancer and other cancers amenable to treatment with IL-33. Building on these developments and discoveries, the present invention provides a variety of new and improved recombinant IL-33 proteins and methods of use of such recombinant IL-33 proteins.

[0017] Thus, in one aspect the present invention provides recombinant IL-33 proteins that comprise a human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain contains one or more mutations as compared to the corresponding wild-type human IL-33 protein sequence. Further details of these recombinant IL-33 proteins, including the mutations contained therein, are provided in subsequent sections of this patent disclosure.Atorney Docket No. MSKCC.064.WO1

[0018] Electronically filed on: January 9, 2026 In another aspect the present invention provides recombinant IL-33 proteins that are Fc fusion proteins comprising a human IL-33 protein domain (“IL-33 domain”) and a human IgG Fc domain (“Fc domain”), wherein the IL-33 domain contains one or more mutations as compared to the corresponding wild-type human IL-33 protein sequence, and wherein, optionally, the Fc domain also contains one or more mutations as compared to the corresponding wild-type human IgGFc sequence. These recombinant IL-33 proteins may be provided in dimeric forms that are either monovalent (each dimer comprising only one IL-33 domain) or bivalent (each dimer comprising two IL-33 domains). Further details of these recombinant IL-33 proteins, including the mutations contained in the IL-33 and Fc domains thereof, and the monovalent and bivalent forms thereof, are provided in more detail in subsequent sections of this patent disclosure.

[0019] In another aspect the present invention provides compositions comprising the recombinant IL-33 proteins described herein. For example, in some embodiments, the present invention provides pharmaceutical compositions comprising a recombinant IL-33 protein as described herein and at least one pharmaceutically acceptable carrier or excipient.

[0020] In another aspect the present invention provides various methods of using the recombinant IL-33 proteins of the present invention.

[0021] For example, in some embodiments, the present invention provides methods of activating IL-33 receptors, such methods comprising contacting IL-33 receptors, in vitro or in vivo, with a recombinant IL-33 protein as described herein.

[0022] In other embodiments, the present invention provides methods of expanding and / or activating ILC2 cells, such methods comprising contacting ILC2 cells, in vitro or in vivo, with a recombinant IL-33 protein as described herein.

[0023] In other embodiments, the present invention provides methods of enhancing the formation of intra-tumoral tertiary lymphoid structures (TLSs) in subjects in need thereof, such methods comprising administering a recombinant IL-33 protein or pharmaceutical composition as described herein to such a subject.

[0024] Similarly, in other embodiments, the present invention provides methods of treating pancreatic ductal adenocarcinoma (PDAC) in subjects in need thereof, such methods comprising administering a recombinant IL-33 protein or pharmaceutical composition as described herein to such a subject.Atorney Docket No. MSKCC.064.WO1

[0025] Electronically filed on: January 9, 2026 These and other aspects and embodiments of the present invention are described in further detail in subsequent sections of this patent disclosure.

[0026] BRIEF DESCRIPTION OF THE FIGURES

[0027] Fig. 1. Schematic representation of certain IL-33 - Fc-fusion protein formats. In the schematics mature IL-33 protein is represented as a large light gray oval and the Fc proteins are represented as smaller dark gray or black ovals. The left hand and middle panels show mature IL-33 (represented as large light gray oval) fused to human IgGl Fc and human IgG4 (smaller dark gray ovals) at the N-terminus (left-hand panel) or C-terminus (middle panel) via a (G3S)3linker. The (G3S)3linker has the amino acid sequence GGGSGGGSGGGS (SEQ ID NO. 67). The righthand panel shows a monovalent IL-33 - Fc-fusion protein engineered using an Fc with knob-in-hole (KiH) mutations. The Fc portions comprise an IgGl backbone (illustrated using dark gray ovals for the hole arm and black ovals for the knob arm) with IL-33 fused to the C-terminus of the knob arm.

[0028] Fig- 2. Identification ofN-linked glycosylation (N-X-S / T) liabilities in IL-33. Comparison of intact mass data for an IL-33 Fc fusion variant pair that differ only at position 173 of IL-33, i.e. native S173 (Bi_Fc0_CS-IL-33 left and middle graphs) vs S173A-mutated (Bi_FcO_ACS-IL-33right-hand graph). The absence of a higher mass species in Bi_Fc0_ACS-IL-33 corresponding to the 47240 Da species in Bi_Fc0_CS-IL-33 is indicative of N171 glycosylation in the later but not former fusion variant.

[0029] Fig.3A-C. Characterization of Disulfide-Containing IL-33 Variants. Fig.3A. Relative expression of GPI-anchored IL-33 variant proteins as assessed by flow cytometry with rabbit anti-Myc mAb. A rabbit mAb isotype control (Rb IgG) was used as a negative control. Fig.3B.

[0030] Relative binding of IL-33 variant proteins to His-tagged ST2 protein as assessed by flow cytometry. Bound ST2 was detected with dye-labeled anti-His antibody. Fig.3C. Functional activity of IL-33 variant proteins in the IL-33 HEK Blue™ cell reporter assay.

[0031] Fig. 4A-B. Purity of disulfide-stabilized IL-33 Fc fusion variants as assessed by SDS-PAGE. Fig. 4A. Non-reducing SDS-PAGE analysis of stabilized fusion variants and non-stabilized Bi_Fcl_Ml-IL-33 reference. Fig. 4B. Reducing SDS-PAGE analysis of stabilized fusion variants and non-stabilized Bi_Fcl_Ml-IL-33 reference.

[0032] Fig. 5. Purity of disulfide-stabilized IL-33 Fc fusion variants as assessed by analytical SEC. SECAtorney Docket No. MSKCC.064.WO1

[0033] Electronically filed on: January 9, 2026 evaluation of higher purity fusion variants (M3-, M14-, M15- and M16-containing) as compared to non-stabilized Bi_Fcl_Ml-IL-33. The main peak for non-stabilized reference is asymmetric, with the tail (marked by red arrow) being consistent with lower molecular weight fragmentation contaminants. All samples had modest levels of high molecular weight impurities, presumably aggregate species, that eluted at shorter retention times.

[0034] Fig- 6. Functional activity of disulfide-stabilized IL-33 Fc fusion variants. Samples were incubated with HEK-Blue™ IL-33 cells at indicated concentrations. Functional activity, leading to secretion of alkaline phosphatase reporter enzyme, was assayed with a colorimetric substrate. Concentrations at which half-maximal signal was observed were calculated and are reported as EC50 values.

[0035] Fig- 7. Time dependent proteolysis of stabilized IL-33 fusion variants by thermolysin. Samples were incubated with thermolysin for the indicated times, inactivated by addition of EDTA, then analyzed by reducing SDS-PAGE. The figure shows the SDS-PAGE gels for the 5 indicated molecules. While the linker region between IL-33 and Fc was highly susceptible in all samples, subsequent rates of degradation of the IL-33 fragment varied by sample. Approximate times to complete disappearance of the IL-33 band is indicated in the table.

[0036] Fig. 8. Serum stability of stabilized IL-33 fusion variants. Samples were incubated in human serum for the indicated times, IL-33-Fc fusion (or Fc-containing fragments) was recovered by Pro-A purification, then analyzed by non-reducing SDS-PAGE. The figure shows the SDS-PAGE gels for the 5 indicated molecules. A serum protein contaminant (~50 kDa) co-purified with IL-33-Fc fusion and can be detected at every time point. The To sample represents test article incubated in PBS at 37°C for 72 h and accordingly lacks the serum protein contaminant.

[0037] Fig- 9. N297 glycan assignment based on intact mass analysis by mass spectrometry (MS). Electrospray ionization liquid chromatography mass spectrometry (ESI / LC / MS) was used to determine the molecular weight (MW) of glycosylated protein species within each indicated sample. Percent abundance of the Man5 species represents the area of the Man5 peak as a fraction of the combined area for all peaks.

[0038] Fig. 10. Productivity and bioactivity of aglycosylated Mo_aFc_M14-IL-33 and Bi_aFc_M14-IL-33 fusion proteins versus their glycosylated counterparts.

[0039] Fig. 11. Serum stability evaluation of Bi_aFc_M14_IL-33, Mo_aFc_M14_IL-33 andAtorney Docket No. MSKCC.064.WO1

[0040] Electronically filed on: January 9, 2026 Bi_Fcl_Ml_IL-33. Samples were incubated in human serum for the indicated times, IL-33-Fc fusion (or Fc-containing fragment) was recovered by Pro-A purification, then analyzed by nonreducing SDS-PAGE. The SDS-PAGE gels for each of the indicated molecules is shown. A serum protein contaminant (~50 kDa) co-purified with IL-33-Fc fusion and can be detected at every time point. The To sample represents test article incubated in PBS at 37°C for 72 h and accordingly lacks the serum protein contaminant.

[0041] Fig. 12A-D. Mo_aFcl_M14-IL-33 and Bi_aFcl_M14-IL-33 stimulates tertiary lymphoid structures (TLSs) and anti-tumor activity in PDAC. Survival (Fig. 12A), tumor growth (Fig. 12B), intratumoral KLRG1+ ILC2 frequency (Fig. 12C), and TLS density (Fig. 12D) in PDAC mice treated with isotype control, mouse rIL33, or equimolar concentration of Mo_aFcl_M14-IL-33 and Bi_aFcl_M14-IL-33 at 0.04 or 1.14 nmol / kg. Data collected at 3-4 weeks (Fig. 12B-D) after tumor implantation, with n > 7 mice per group with consistent results, n = individual tumors from individual mice analyzed separately. Horizontal bars = median. P values by two-way ANOVA with Tukey’s multiple comparison (c and d).

[0042] Fig. 13A-E. Mo_aFc_M14-IL-33 (H-rIL33-FC) stimulates TLSs and anti-tumor activity in PDAC. Experimental schematic (Fig. 13A), survival (Fig. 13B), tumor growth (Fig. 13C), Intratumoral KLRG1+ ILC2 frequency (Fig. 13D), and TLS density (Fig. 13E) in PDAC mice treated with isotype control, mouse rIL33, or escalating Mo_aFc_M14-IL-33. Data collected at 3-4 weeks (Fig. 13C-E) after tumor implantation, pooled from > 2 independent experiments with n > 3 mice per group with consistent results, n = individual tumors from individual mice analyzed separately. Horizontal bars = median. P values by two-way ANOVA with Tukey’s multiple comparison (Fig. 13D).

[0043] Fig. 14. Elimination of N-linked glycosylation motifs does not affect IL-33 bioactivity. IL-33 bioactivity comparison by HEK-blue report assay of IL-33 mutants (SI 73 A or SI 73 A and C259A respectively named “Bi_Fc0_M0-IL-33 and Bi_Fcl_Ml-IL-33”) leading to elimination of N-linked glycosylation. No significant differences in IL-33 bioactivity were observed between glycosylated IL-33 (Bi_Fc0_CS-IL-33) and variants that with N-linked glycosylation site removed. The ECso for Bi_Fc0_CS-IL-33 was 0.040 nM. The EC50 for Bi_Fcl_Ml-IL-33 was 0.052 nM. The EC50 for Bi_Fc0_M0-IL-33 was 0.071 nM. Bi_Fcl_Ml-IL-33 construct was selected for further development.Atorney Docket No. MSKCC.064.WO1

[0044] Electronically filed on: January 9, 2026 Fig. 15. Fragmentation of Bi_Fcl_Ml-IL-33 Detected by Mass Spectrometry. Figure shows UV trace (upper panel) and total ion current (TIC) trace (lower panel) from LC / MS analysis of Bi_Fcl_Ml-IL-33, demonstrating fragmentation impurities.

[0045] Fig. 16. Fragmentation of Bi_Fcl_Ml-IL-33 in serum. The figure shows the serum stability time course of recombinant Bi Fcl Ml -IL-33. Post serum incubation, Bi_Fcl _Ml-IL-33 fusion was recaptured by Pro-A chromatography and purified material was analyzed by SDS-PAGE under reducing (left panel) and non-reducing (right panel) conditions. The results show that Bi_Fcl_Ml-IL-33 is susceptible to proteolysis.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] Some aspects and embodiments of the present invention are described in the Summary of the Invention, Brief Description of the Figures, Examples, Claims, and Figures sections of this patent disclosure. This Detailed Description of the Invention section provides additional embodiments, and additional description and details relating to aspects and embodiments described throughout the present patent disclosure, and is intended to be read in conjunction with all other sections of the present patent disclosure.

[0048] For ease of reference, this Detailed Description of the Invention section is arranged using certain headings and subheadings. These headings and subheadings are not intended to limit the scope of the invention in any way. All sections of this patent disclosure should be read in conjunction with all other sections of the patent disclosure. Furthermore, one of ordinary skill in the art will understand that the various aspects and embodiments of the present invention can be combined in various different ways. All such combinations fall within the scope of the present invention.

[0049] Definitions & Abbreviations

[0050] In order that the present invention can be more readily understood, certain terms are defined below. Additional definitions are set forth throughout the disclosure where the terms are used. Unless defined otherwise, or a specific meaning is clear from the context of use, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related. The Dictionary of Cell and Molecular Biology (5th ed. J.M. Lackie ed., 2013), the Oxford Dictionary of Biochemistry and Molecular Biology (2d ed. R. Cammack et al. eds., 2008), and The Concise Dictionary of Biomedicine and Molecular Biology (2d ed. P-S. Juo, 2002) can provide one of skill with general definitions ofAtorney Docket No. MSKCC.064.WO1

[0051] Electronically filed on: January 9, 2026 some of the terms used herein.

[0052] Units, prefixes, and symbols as used herein are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges provided herein are inclusive of the numbers defining the range.

[0053] As used herein, the singular forms “a,” “an,” and “the” include plural referents, unless the context clearly dictates otherwise. The terms “a” (or “an”) as well as the terms “one or more” and “at least one” can be used interchangeably.

[0054] As used herein, “and / or ’ is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone).

[0055] As used herein the terms “about” and “approximately,” when used in relation to numerical values, mean within + or - 10% of the stated value.

[0056] As used herein, the term “agonist” refers to a molecule (e.g., a recombinant IL-33 protein) with the ability to produce a detectable, measurable, or observable activation of a specified receptor (e.g., an IL-33 receptor), for example in an in vitro or in vivo assay.

[0057] Amino acids are typically referred to using either their accepted three-letter or one-letter symbols as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids are referred to herein using their one-leter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0058] As used herein the term “bivalent” refers to a recombinant IL-33 protein that is a dimer and each monomer constituting the dimer comprises an IL-33 domain.

[0059] The terms “comprising,” “consisting of’, and / or “consisting essentially of’ are used herein consistently with their accepted meanings according to U.S. patent law. Wherever embodiments are described with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of’, and / or “consisting essentially of,” are also intended and included within the scope of the invention.

[0060] As used herein, the abbreviation “IL” refers to interleukin and the abbreviations “IL-33” andAtorney Docket No. MSKCC.064.WO1

[0061] Electronically filed on: January 9, 2026 “IL33” refer to interleukin 33. The abbreviations IL-33 and IL33 may be used interchangeably herein.

[0062] As used herein, the term “effective amount,” when used in relation to the recombinant IL-33 proteins of the present invention, refers to an amount of a recombinant IL-33 protein that is sufficient to have activity as an agonist of an IL-33 receptor, or to achieve detectable, measurable, or observable agonism of an IL-33 receptor, or to achieve a detectable, measurable, or observable biological effect or treatment effect (see definition of “treatment” below defined below), in the stated situation. The amount of a given recombinant IL-33 protein, or composition comprising such a recombinant IL-33 proteins, that is an “effective amount” may depend on variety of factors including, but not limited to, the species of the subject (e.g., whether a human or some other animal species), the age of the subject, the sex of the subject, the weight of the subject, the formulation of the recombinant IL-33 protein, the route of administration of the recombinant IL-33 protein, the planned dosing regimen for the recombinant IL-33 protein, etc. The effective amount (which may be a range of effective amounts) can be determined by standard techniques, for example using in vitro assays and / or in vivo assays in the intended subject species or any suitable animal model species. Suitable methods for determining an effective amount include, but are not limited to, dose-escalation studies, extrapolation from doseresponse curves, and / or extrapolation from other data derived from in vitro and / or in vivo model systems. In some embodiments, the effective amount may be determined according to the judgment of a medical or veterinary practitioner based on the specific circumstances.

[0063] As used herein, the terms “exemplary” or “e.g.” or “for example” or “such as” mean serving as an example, instance, or illustration. These terms are non-limiting and are not intended to denote preferred embodiments.

[0064] The abbreviation “Fc” refers to a "fragment crystallizable" region / domain of, or derived from, the carboxy -terminal portion of the heavy chain of an antibody or immunoglobulin molecule, such as from an IgG molecule. The term “Fc” is used herein consistently with its accepted meaning in the art.

[0065] As used herein, the term “isolated” refers to proteins that are not within a subject or within a cell (such as a host cell used in manufacturing) and are typically in a form not found in nature. In some embodiments, an isolated protein may have been purified to a degree that it is not in a form in which it is found in nature. It is to be understood that the present invention provides isolatedAtorney Docket No. MSKCC.064.WO1

[0066] Electronically filed on: January 9, 2026 versions of each of the recombinant IL-33 proteins, IL-33 domains, and Fc domains described herein.

[0067] As used herein the term “monovalent” refers to a recombinant IL-33 protein that is a dimer in which only one of the two monomers constituting the dimer comprises an IL-33 domain.

[0068] As used herein, the term “protein” refers to a polymer of amino acids linked via peptide bond(s). Proteins may be represented herein by their amino acid sequence. Such amino acid sequences may be referred to using a sequence identification number (SEQ ID NO:).Unless stated otherwise, proteins represented by their amino acid sequence are presented with the N-terminus on the left and the sequence is writen from the N-terminus to the C-terminus.

[0069] As used herein, the term “pharmaceutical composition” refers to a composition that is in such form as to permit the biological activity of the active agent(s) contained therein (e.g., the recombinant IL-33 proteins described herein), and that contain no additional components that are unacceptably toxic to a subject to which the composition may be administered. In some embodiments, such compositions may be sterile.

[0070] As used herein, the term “subject” refers to an individual for whom treatment using a recombinant IL-33 protein or method as described herein is desired, needed or performed. As used herein, a subject “in need thereof’ is a subject who could reasonably be expected to benefit from treatment using a recombinant IL-33 protein, pharmaceutical composition, or method as described herein - for example as determined by a medical professional. In some embodiments, the subject is any mammalian subject, including, but not limited to, humans (male and / or female), non-human primates, dogs, cats, rodents (such as rats, mice and guinea pigs), cows, animals used in husbandry, animals kept as pets, animals kept and in zoos, and animals used in preclinical testing. In preferred embodiments, the subject is a human.

[0071] As used herein, the terms “treat,” “treating,” and “treatment” refer to achieving, and / or performing a method that achieves, a detectable, measurable, or observable improvement in one or more clinical parameters or symptoms of the stated condition. For example, in embodiments where the stated condition is pancreatic cancer (e.g., pancreatic ductal adenocarcinoma or “PDAC”), the terms “treat,” “treating,” and “treatment” encompass achieving, and / or performing a method that achieves, a detectable improvement in one or more clinical indicators or symptoms associated with pancreatic cancer (e.g. PDAC) such as: reducing the rate of growth of a pancreatic tumor (or of pancreatic tumor cells), halting the growth of a pancreatic tumor (or of pancreatic tumor cells),Atorney Docket No. MSKCC.064.WO1

[0072] Electronically filed on: January 9, 2026 causing regression of a pancreatic tumor (or of pancreatic tumor cells), reducing the size of a pancreatic tumor (for example as measured in terms of tumor volume or tumor mass), reducing the grade of a pancreatic tumor, eliminating a pancreatic tumor (or pancreatic tumor cells), preventing, delaying, or slowing recurrence (rebound) of a pancreatic tumor, improving symptoms associated with a pancreatic tumor, improving survival from a pancreatic tumor, inhibiting or reducing spreading of a pancreatic tumor (e.g. metastases), and the like. In each of the embodiments described herein that refer to a method of treatment, a method of achieving any one or more of the specific parameters listed above is also contemplated. For example, for each of the embodiments described herein that refers to a method of treating pancreatic cancer (e.g. PDAC), the following methods are also contemplated and are intended and fall within the scope of the invention: (a) a method of reducing the rate of growth of a pancreatic tumor (or of pancreatic tumor cells), (b) a method of halting the growth of a pancreatic tumor (or of pancreatic tumor cells), (c) a method of causing regression of a pancreatic tumor (or of pancreatic tumor cells), (d) a method of reducing the size of a pancreatic tumor (for example as measured in terms of tumor volume or tumor mass), (e) a method of reducing the grade of a pancreatic tumor, (f) a method of eliminating a pancreatic tumor (or pancreatic tumor cells), (g) a method of preventing, delaying, or slowing recurrence (rebound) of a pancreatic tumor, (h) a method of improving symptoms associated with a pancreatic tumor, (i) a method of improving survival from a pancreatic tumor, and (j) a method of inhibiting or reducing spreading of a pancreatic tumor (e.g. metastasis).

[0073] The term “means” is used herein consistently with its usage in U.S. patent law, and shall be given its broadest possible interpretation in accordance with 35 U.S.C. section 112(f).

[0074] Accordingly, a claim incorporating the term “means” shall cover all structures, materials, or acts described herein, and all equivalents thereof. Further, the structures, materials, or acts shall include all of those described in the Summary of the Invention, Brief Description of the Figures, Figures, Detailed Description of the Invention, Examples, Claims, Abstract, and Sequence Listing. It is intended that the recombinant IL-33 proteins of the present invention may also be referred to as a “means” for achieving a function or effect as described herein, such as a means for activating an IL-33 receptor, a means for expanding or activating ILC2 cells, a means for inducing intratumoral tertiary lymphoid structures, and / or a means for treating pancreatic ductal adenocarcinoma (PDAC). Thus, whenever an embodiment of the present invention is described without using “means” language, it is to be understood that an analogous embodiment, described as a “means” for achieving a function or effect that is described herein, is also contemplated and falls within the scope of the present invention.Atorney Docket No. MSKCC.064.WO1

[0075] Electronically filed on: January 9, 2026 The term “variant” as used herein refers to a protein or protein domain that differs from a specified comparator protein or protein domain (e.g., a wild-type IL-33 protein or a wild-type Fc domain) by having one or more amino acid substitutions, insertions, or deletions as compared thereto. One of skill in the art will appreciate that variants of many of the specific amino acid sequences described herein can be used provided that the variant retains the desired or specified function(s), such as, for example, IL-33 activity and / or ST2 binding in the case of IL-33 proteins / domains and structural stability and / or enhanced half-life and / or interaction with the neonatal Fc receptor (FcRn) in the case of Fc proteins / domains.

[0076] Other terms are defined elsewhere in this patent disclosure, or else are used in accordance with their accepted meaning in the art.

[0077] Recombinant IL-33 Proteins

[0078] As summarized above, the present invention involves novel recombinant IL-33 proteins that were developed as a result of extensive efforts undertaken to engineer mature human IL-33 to: (a) stabilize the IL-33 molecule using disulfide bonds introduced at sites selected based on crystal structure analysis, (b) eliminate sites susceptible to unpaired cysteine oxidation, (c) remove glycosylation sites, and / or (d) fuse the IL-33 molecule to an IgG Fc domain. As described in the Examples section of this patent disclosure, the recombinant IL-33 proteins produced exhibit enhanced stability, a longer half-life, resistance to cysteine oxidation and proteolysis, native-like activation of the ST2 / IL-lRAcP pathway, induction of ILC2 cell expansion and activation, enhanced formation of intra-tumoral tertiary lymphoid structures (TLSs), and effective control tumor growth control in a preclinical pancreatic cancer model. Further details of these novel recombinant IL-33 proteins are described below.

[0079] IL-33 Domains

[0080] As summarized above, in one aspect the present invention provides recombinant IL-33 proteins that comprise a human IL-33 protein domain (“IL-33 domain”), while in another aspect the present invention provides recombinant IL-33 proteins that are Fc fusion proteins comprising a human IL-33 protein domain (“IL-33 domain”) and a human IgGFc domain (“Fc domain”). In each aspect, and in each of the embodiments of the present invention, the IL-33 domain comprises a variant of the mature wild-type human IL-33 protein that has one or more mutations as compared to the mature wild-type human IL-33 sequence.Atorney Docket No. MSKCC.064.WO1

[0081] Electronically filed on: January 9, 2026 In nature, the full-length human wild-type human IL-33 is the 270 amino acid unprocessed form of human IL-33 that is found within cells, while mature wild-type human IL-33 is the 159 amino acid processed form of human IL-33 produced following extracellular release and proteolytic cleavage of the full-length form. Mature human IL-33, which in nature constitutes amino acids 112-270 of the full-length form, is much more biologically potent than full length IL-33 and contains the cytokine domain that activates the ST2 receptor. Table A below provides the amino acid sequences of the full-length (SEQ ID NO. 1) and mature (SEQ ID NO. 2) forms of wildtype human IL-33.

[0082] Table A

[0083] &

[0084]

[0085] The IL-33 domains present in the recombinant IL-33 proteins of the present invention comprise variants of the mature wild-type human IL-33 protein (SEQ ID NO. 2) that comprise one or more mutations as compared to the mature wild-type human IL-33 sequence. It should be noted that, while the mature form of IL-33 that is produced in nature is the 159 amino acid form consisting of amino acid residues 112-270 of the full-length form, the mature wild-type human IL-33 protein / domain present in the recombinant IL-33 proteins of the present invention need not include all 159 amino acids. For example, shorter versions of mature wild-type human IL-33 that retain IL-33 activity but are truncated may be used. For example, the mature wild-type human IL-33 used may be truncated by 1, 2, 3, 4, 5, or 6 amino acids at the N-terminus and / or by 1, 2, 3, or 4 amino acids at the C-terminus. For example, the mature wild-type human IL-33 used may have an N-terminus that commences at any amino acid between residues 112-118 and may have a C-terminus that commences at any amino acid between residues 266-270. By way of further example, the mature wild-type human IL-33 used may may comprise amino acids 112-270, 113-270, 114-270, 115-270, 116-270, 117-270, 118-270, 112-269, 113-269, 114-269, 115-269, 116-269, 117-269, 118-269, 112-268, 113-268, 114-268, 115-268, 116-268, 117-268, 118-Atorney Docket No. MSKCC.064.WO1

[0086] Electronically filed on: January 9, 2026 268, 112-267, 113-267, 114-267, 115-267, 116-267, 117-267, 118-267, 112-266, 113-266, 114- 266, 115-266, 116-266, 117-266, or 118-266 of the full length IL-33 protein.

[0087] Specific mutations are described herein by specifying, for a given amino acid position, the amino acid residue present in the wild-type IL-33 protein and the substituted amino acid present in its place. For example, the notation SI 73 A refers to a mutation in which the amino acid serine (S) at the amino acid position that corresponds to amino acid number 173 of wild-type human IL-33 is substituted for the amino acid alanine (A), wherein the amino acid positions are numbered using the numbering of the full length wild-type human IL-33 protein having Uniprot ID: 095760, which is 270 amino acids long and has the amino acid sequence shown in Table A above (SEQ ID NO. 1).

[0088] Accordingly, in one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises one or more mutations as compared to the mature wild-type human IL-33 sequence, wherein the mutations are selected from those listed in Table C. The functional significance of each of these mutations, and examples of molecules containing various combinations of these mutations, are further described in the Examples section of this patent disclosure as well as in Tables B and C below.

[0089] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a S173A mutation.

[0090] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S or C208A mutation, a C227S or C227A mutation, a C232S or C232A mutation, and a C259S or C259A mutation. In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S or C208A mutation, a C227S or C227A mutation, and a C259S or C259A mutation, but does not comprise a mutation at C232.

[0091] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227S mutation,Atorney Docket No. MSKCC.064.WO1

[0092] Electronically filed on: January 9, 2026 a C232S mutation, and a C259S mutation. In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227S mutation, and a C259S mutation, but does not comprise a mutation at C232.

[0093] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227S mutation, a C232S mutation, and a C259A mutation. In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227S mutation, and a C259A mutation, but does not comprise a mutation at C232.

[0094] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation. In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises a C208S mutation, a C227A mutation, and a C259A mutation, but does not comprise a mutation at C232.

[0095] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises at least one pair of cysteines that create an intramolecular disulfide linkage, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: Pl 18C and S162C; L123C and K158C; S127C and K266C; Y129C and L264C; I134C and V147C; F136C and D157C; V147C and V242C; L161C and V184C; S162C and T185C; Y164C and M183C; S166C and M183C; S166C and V219C; H168C and V219C; S170C and G179C; N171C and G179C; A196C and Q215C; A196C and C232; V203C and I240C; V203C and L247C; Q215C and T234C; S229C and I263C; F239C and I263C; G241C and N262C; and G241C and I263C. In some embodiments theAtorney Docket No. MSKCC.064.WO1

[0096] Electronically filed on: January 9, 2026 IL-33 domain comprises at least two pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least two disulfide linkage sites. In some embodiments the IL-33 domain comprises at least three pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least three disulfide linkage sites. In some embodiments the IL-33 domain comprises at least four pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least four disulfide linkage sites.

[0097] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: L123C and K158C; Y129C and L264C; F136C and D157C; V147C and V242C; S166C and M183C; H168C and V219C; S170C and G179C; N171C and G179C; A196C and C232; Q215C and T234C; G241C andN262C; and G241C and I263C. In some embodiments the IL-33 domain comprises at least two pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least two disulfide linkage sites. In some embodiments the IL-33 domain comprises at least three pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least three disulfide linkage sites. In some embodiments the IL-33 domain comprises at least four pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least four disulfide linkage sites.

[0098] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: L123C and K158C; H168C and V219C; S170C and G179C; and N171C and G179C. In some embodiments the IL-33 domain comprises at least two pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least two disulfide linkage sites. In some embodiments the IL-33 domain comprises at least three pairs of cysteines selected from those listed above such that the IL-33 domain comprises at least three disulfide linkage sites. In some embodiments the IL-33 domain comprises all four pairs of cysteines selected from those listed above such that the IL-33 domain comprises four disulfide linkage sitesAtorney Docket No. MSKCC.064.WO1

[0099] Electronically filed on: January 9, 2026 In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: H168C and V219C; and S170C and G179C. In some embodiments the IL-33 domain comprises both of the pairs of cysteines listed above such that the IL-33 domain comprises two disulfide linkage sites.

[0100] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is H168C and V219C.

[0101] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises: (a) a S173A mutation, and (b) a C208S mutation, a C227S mutation, a C232S mutation, and a C259A mutation.

[0102] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises: (a) a S173A mutation, and (b) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation.

[0103] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises:

[0104] (a) a SI 73 A mutation,

[0105] (b) either (i) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, or (ii) a C208S mutation, a C227A mutation, and a C259A mutation, and

[0106] (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteineAtorney Docket No. MSKCC.064.WO1

[0107] Electronically filed on: January 9, 2026 mutations: Pl 18C and S162C; L123C and K158C; S127C and K266C; Y129C and L264C; I134C and V147C; F136C and D157C; V147C and V242C; L161C and V184C; S162C and T185C; Y164C and M183C; S166C and M183C; S166C and V219C; H168C and V219C; S170C and G179C; N171C and G179C; A196C and Q215C; A196C and C232; V203C and I240C; V203C and L247C; Q215C and T234C; S229C and 1263 C; F239C and I263C; G241C and N262C; and G241C and I263C.

[0108] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises:

[0109] (a) a SI 73 A mutation,

[0110] (b) either (i) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, or (ii) a C208S mutation, a C227A mutation, and a C259A mutation, and

[0111] (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: L123C andK158C; Y129C and L264C; F136C and D157C; V147C and V242C; S166C and M183C; H168C and V219C; S170C and G179C; N171C and G179C; A196C and C232; Q215C and T234C; G241C and N262C; and G241C and 1263 C.

[0112] In some embodiments the IL-33 domain comprises at least two pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises at least two disulfide linkage sites. In some embodiments the IL-33 domain comprises at least three pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises at least three disulfide linkage sites. In some embodiments the IL-33 domain comprises at least four pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises at least four disulfide linkage sites.

[0113] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises:

[0114] (a) a SI 73 A mutation,Atorney Docket No. MSKCC.064.WO1

[0115] Electronically filed on: January 9, 2026 (b) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, and

[0116] (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: L123C and K158C; H168C and V219C; S170C and G179C; andN171C and G179C.

[0117] In some embodiments the IL-33 domain comprises at least two pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises at least two disulfide linkage sites. In some embodiments the IL-33 domain comprises at least three pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises at least three disulfide linkage sites. In some embodiments the IL-33 domain comprises all four pairs of cysteines selected from those listed in part (c) above such that the IL-33 domain comprises four disulfide linkage sites.

[0118] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises:

[0119] (a) a SI 73 A mutation,

[0120] (b) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, and

[0121] (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is selected from the following paired cysteine residues / cysteine mutations: H168C and V219C; and S170C and G179C.

[0122] In some embodiments the IL-33 domain comprises both pairs of cysteines listed in part (c) above such that the IL-33 domain comprises least two disulfide linkage sites.

[0123] In one embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises a variant of mature wild-type human IL-33 protein (e.g., SEQ ID NO. 2) that comprises:

[0124] (a) a SI 73 A mutation,Atorney Docket No. MSKCC.064.WO1

[0125] Electronically filed on: January 9, 2026 (b) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, and

[0126] (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation, and wherein the at least one pair of cysteines is H168C and V219C.

[0127] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 4-30 (IL-33 constructs ASC-IL-33 and MO-IL-33 to M25-IL-33), as provided in Table B below.

[0128] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 6-30 (IL-33 constructs Ml -IL-33 to M25-IL-33) as provided in Table B below.

[0129] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 7-30 (IL-33 constructs M2 -IL-33 to M25-IL-33) as provided in Table B below.

[0130] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 8, 10, 12, 13, 17, 19, 20, 21, 23, 26, 29 and 30 (IL-33 constructs M3-IL-33, M5-IL-33, M7-IL-33, M8-IL-33, M12-IL-33, M14-IL-33, M15-IL-33, M16-IL-33, M18-IL-33, M21-IL-33, M24-IL-33, M25-IL-33) as provided in Table B below.

[0131] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 8, 19, 20, and 21 (IL-33 constructs M3-IL-33, M14-IL-33, M15-IL-33, and M16-IL-33) as provided in Table B below. Each of these IL-33 domains comprises: (a) a S173A mutation, (b) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, and (c) at least one pair of cysteines that create a disulfide linkage site, wherein at least one cysteine in each pair of cysteines is a “to cysteine” mutation.Attorney Docket No. MSKCC.064.WO1

[0132] Electronically filed on: January 9, 2026 In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 19 and 20 (IL-33 constructs M14-IL-33 and M15-IL-33) as provided in Table B below.

[0133] In another embodiment the present invention provides a recombinant IL-33 protein that comprises at least one human IL-33 protein domain (“IL-33 domain”), wherein the IL-33 domain comprises the amino acid sequence of SEQ ID NO. 19 (IL-33 construct M14-IL-33) as provided in Table B below.

[0134] Table B: Amino Acid Sequences of IL-33 Domains

[0135]

[0136] Atorney Docket No. MSKCC.064.WO1

[0137] Electronically filed on: January 9, 2026

[0138]

[0139] Atorney Docket No. MSKCC.064.WO1

[0140] Electronically filed on: January 9, 2026

[0141]

[0142] Table C below lists the mutations present in each of the IL-33 domains for which the amino acid sequences are provided in Table B.

[0143] Table C: Mutations in IL-33 Domains

[0144]

[0145] Atorney Docket No. MSKCC.064.WO1

[0146] Electronically filed on: January 9, 2026

[0147]

[0148] In Table C above, theAsymbol indicates that no mutation (amino acid substitution) was made at amino acid position 232 in variant Ml 8-IL-33, thus variant M18-IL-33 retains the wild-type cysteine (C) residue at amino acid residue 232.

[0149] It should be noted that, while much of the present patent disclosure relates to recombinant IL-33 proteins that are Fc fusion proteins comprising both an IL-33 domain and an Fc domain, the novel IL-33 domains presented herein may be useful in other contexts, including alone (in which case they may be useful in any other situation in which a mature IL-33 protein is useful) or in conjunction with other components. For example, the IL-33 domains of the present invention may be modified by addition of various half-life increasing moieties (such as PEG), other fusion partners (such as albumin binding domains, and the like), tags useful for protein detection, tags useful for protein purification, components useful for the targeting of therapeutic agents, radiolabels, and the like.

[0150] Fc Domains

[0151] As summarized above, in one aspect the present invention provides recombinant IL-33 proteins that are Fc fusion proteins comprising: (a) at least one human IL-33 protein domain (“IL-33Atorney Docket No. MSKCC.064.WO1

[0152] Electronically filed on: January 9, 2026 domain”), as described in section (i) above, and (b) at least one human IgG Fc domain (“Fc domain”).

[0153] In some embodiments, any suitable Fc domain can be used in the Fc fusion proteins of the present invention. For example, in some embodiments any Fc domain that has been mutated or modified to reduce or eliminate Fc effector function can be used in the Fc fusion proteins of the present invention. Numerous Fc mutations and Fc modifications are known in the art that can be used to reduce or eliminate Fc effector function (in an IgGl Fc or other IgG Fes), including various point mutations and Fc molecules containing hybrid Fc regions. For example, inclusion of an IgG2 hinge region in an IgGl Fc can be used to reduce or eliminate IgGl Fc effector function. In some embodiments, an Fc containing any Fc mutation and / or Fc modification that reduces or eliminates Fc effector function that is known in the art may be used in the IL-33-Fc fusion proteins of the present invention. Examples of such Fc mutations and / or modifications that can be used in accordance with the present invention are described in Hale et al., “Systematic analysis of Fc mutations designed to reduce binding to Fc-gamma receptors.” MAbs. 2024 Jan-Dec;16(l):2402701, PMID: 39279104, the contents of which are hereby incorporated by reference.

[0154] Examples of certain Fc domains and Fc mutations that can be used in the Fc fusion proteins of the present invention are described below in this section (ii) and in Table E, below. (Details of the fusion proteins comprising both an IL-33 domain and an Fc domain are described in section (iii) below).

[0155] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is, or is a variant of, the wild-type Fc region of a human IgGl, IgG2, IgG3, or IgG4 protein.

[0156] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the wild-type Fc region of a human IgGl, IgG2, IgG3, or IgG4 protein that has been mutated or modified to reduce or eliminate Fc effector function.

[0157] In embodiments where the Fc domain is a variant of the wild-type Fc, the Fc domain comprises one or more mutations (specifically amino acid substitutions) as compared to the Fc region of the corresponding wild-type human IgG Fc. Examples of such variants are described herein by specifying, for a given amino acid position, the amino acid residue present in the wild-type protein and the substituted amino acid present in the variant protein. For example, the notation L234A refers to a mutation that is a substitution of the amino acid leucine (L) at amino acidAtorney Docket No. MSKCC.064.WO1

[0158] Electronically filed on: January 9, 2026 position 234 in the wild-type Fc for the amino acid alanine (A) in the variant Fc, wherein the amino acid positions are numbered using the Eu constant region numbering scheme as described in Edelman et al., “The covalent structure of an entire gammaG immunoglobulin molecule.” Proc Natl Acad Sci U S A. 1969 May;63(l):78-85. PMID: 5257969.

[0159] In those embodiments that involve an Fc domain(s) that is, or that is a variant of, the Fc region of human IgGl, in some embodiments the Fc domain may comprise amino acids 221-447 (using the Eu constant region numbering scheme) of human IgGl, or may comprise a variant of amino acids 221-447 of human IgGl to the extent that the Fc domain contains one or more amino acid substitutions as compared to the wild-type human IgGl protein.

[0160] In those embodiments that involve an Fc domain(s) that is, or that is a variant of, the Fc region of human IgG4, in some embodiments the Fc domain may comprise amino acids 216-447 (using the Eu constant region numbering scheme) of human IgG4, or may comprise a variant of amino acids 216-447 of human IgG4 to the extent that the Fc domain contains one or more amino acid substitutions as compared to the wild-type human IgG4 protein.

[0161] Table D below provides the amino acids sequences of amino acids 221-447 of a wild-type human IgGl, and amino acids 216-447 of a wild-type human IgG4.

[0162] Table D

[0163]

[0164] It should be noted that there are multiple allotypes in humans such that there is more than one “wild type” human IgGl Fc, IgG2 Fc, IgG3 Fc and IgG4 Fc sequence.

[0165] SEQ ID NO. 31 provided in Table D above is the Fc amino acid sequence of the IGHGl*01 allotype of wild-type human IgGl, which is also known as Glm(za) or Glml . While many of the mutations described herein were made using the Fc domain of the IGHGl*01 allotype ofAtorney Docket No. MSKCC.064.WO1

[0166] Electronically filed on: January 9, 2026 wild-type human IgGl (SEQ ID NO. 31), the same mutations can be made in the Fc domains of other allotypes of wild-type human IgGl . All such mutants are encompassed herein.

[0167] Similarly, SEQ ID NO. 32 provided in Table D above is the Fc amino acid sequence of the IGHG4*01 allotype of wild-type human IgG4, which is also known as G4m(a). While many of the mutations described herein were made within the Fc domain of the IGHG4*01 allotype of wild-type human IgG4 (SEQ ID NO. 32), the same mutations can be made in the Fc domains of other allotypes of wild-type human IgG4. All such mutants are encompassed herein.

[0168] In some embodiments the Fc domain(s) is, or is a variant of, the Fc region of a wild-type human IgGl protein (for example, that having the amino acid sequence of SEQ ID NO. 31) or a wildtype human IgG4 protein (for example, that having the amino acid sequence of SEQ ID NO. 32).

[0169] In some embodiments the Fc domain(s) is a variant of the Fc region of a wild-type human IgGl protein (for example, that having the amino acid sequence of SEQ ID NO. 31).

[0170] In some embodiments the Fc domain(s) is a variant of the Fc region of a wild-type human IgG4 protein (for example, that having the amino acid sequence of SEQ ID NO. 32).

[0171] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises one or more of the following mutations: L234A, L235A, P33 IS, and / or N297G. These mutations reduce or eliminate potential Fc effector function. As described above, while this embodiment utilizes L234A, L235 A, P331 S, and / or N297G mutations, alternative Fc mutations and modifications known in the art can also be used to reduce or eliminate potential Fc effector function in an IgGl Fc.

[0172] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises each of the following mutations: L234A, L235A, P33 IS, and N297G.

[0173] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises each of the following mutations: L234A, L235A, and P33 IS.

[0174] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of a Fc region of the wild-type human IgGl protein, wherein the Fc domain comprisesAtorney Docket No. MSKCC.064.WO1

[0175] Electronically filed on: January 9, 2026 each of the following mutations: L234A and L235A.

[0176] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises an N297G mutation.

[0177] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises an N297G mutation and does not comprise an L234A, L235A, or P331 S mutation.

[0178] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgGl protein, wherein the Fc domain comprises an N297G mutation and does not comprise a L234A mutation, does not comprise a L235A mutation, and does not comprise a P33 IS mutation.

[0179] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a wild-type human IgG4 protein, wherein the Fc domain comprises one or more of the following mutations: F234A, L235A, and / or S228P. The F234A and L235A mutations reduce or eliminate potential Fc effector function. As described above, while this embodiment utilizes the F234A and L235A mutations, alternative Fc mutations and modifications known in the art can also be used to reduce or eliminate potential Fc effector function in an IgG4 Fc. For example, in some embodiments an N297G can be used in place of the F234A and L235A mutations to reduce or eliminate IgG4 Fc effector function.

[0180] As described in further detail in section (iii) below, the Fc fusion proteins of the present invention dimerize. The dimeric Fc fusion proteins may be either monovalent or bivalent. In the monovalent Fc fusion proteins, only one of the two Fc chains in the dimer comprises an IL-33 domain. In the bivalent Fc fusion proteins, each of the two Fc chains in the dimer comprises an IL-33 domain. Fig. 1 provides a schematic representation of such monovalent and bivalent Fc fusions proteins. Both the monovalent and bivalent Fc fusion proteins of the present invention can comprise any of the Fc mutations described above (i.e., in the case of IgGl Fc fusions, any one of more of the L234A, L235A, P33 IS, and / or N297G mutations, and in the case of IgG4 Fc fusions, any one of more of the F234A, L235A, and / or S228P mutations) in any of the combinations described above. The monovalent Fc fusion proteins of the present invention may also comprise one or more additional mutations to promote heterodimerization of the two different Fc chains (i.e., the chain with the IL-33 domain and the chain without the IL-33Attorney Docket No. MSKCC.064.WO1

[0181] Electronically filed on: January 9, 2026 domain), such as the well-known “knob-in-hole” or “KiH” mutations. The "knob" mutations result in a small structural change on one Fc chain that fits into a complementary "hole" on the other chain, ensuring that the different chains preferentially heterodimerize instead of forming homodimers. The Fc chain that contains the knob mutation may be referred to herein as the “knob chain” or the “knob arm” and the Fc chain that contains the hole mutation may be referred to herein as the “hole chain” or the “hole arm.”

[0182] Accordingly, in some embodiments an Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a human IgGl protein that comprises a T336W “knob” mutation, and as such may be referred to as a “knob arm” or “knob chain.”

[0183] Similarly, in some embodiments an Fc domain(s) of an Fc fusion protein of the present invention is a variant of the Fc region of a human IgGl protein that comprises each of the T366S, L368A, and Y407V “hole” mutations, and as such may be referred to as a “hole arm” or “hole chain.” In some such embodiments the hole chain also comprises a H435R mutation, which reduces binding to Protein A and Protein A resins used in protein purification thereby simplifying removal of any homodimeric hole arm contaminants.

[0184] Table E: Mutations in Fc Domains

[0185]

[0186] Atorney Docket No. MSKCC.064.WO1

[0187] Electronically filed on: January 9, 2026 In Table E the location and identity of substitutions introduced in human IL-33 are described using the amino acid numbering of full-length human IL-33 protein (Uniprot ID: 095760, i.e., SEQ ID NO. 1). In Table E the location and identity of substitutions in IgGl and IgG4 Fc are described using the amino acid numbering of the Eu constant region numbering scheme as described in Edelman et al., “The covalent structure of an entire gammaG immunoglobulin molecule.” Proc Natl Acad Sci U S A. 1969 May;63(l):78-85. PMID: 5257969. In Table E the Bi_ vs Mo_ prefixes refer to bivalent (homodimeric) vs monovalent (heterodimeric) Fc fusions. In Table E fusion proteins are named as a composite of the constituent IL-33 and Fc components, with fusion orientation defined by the ordering of “Fc” and “IL-33” within a given construct name (e.g. IL-33_Bi_Fc0 and Bi_Fc0_IL-33 are fusions of wild type IL-33 to the N- and C-terminus, respectively, of IgGl (L234A / L235A) Fc.

[0188] In some embodiments the Fc domain(s) of an Fc fusion protein of the present invention is the Fc domain portion present in the Fc fusion proteins of any one of SEQ ID NOs. 33-65, as presented in Table E in section (iii) below.

[0189] In some embodiments the Fc domain(s) of a bivalent Fc fusion protein of the present invention is the Fc domain portion present in the Fc fusion proteins of any one of SEQ ID NOs 33-49, 52, 55, 58 and 61-65, as presented in Table E in section (iii) below.

[0190] In some embodiments the Fc domain(s) of a monovalent Fc fusion protein of the present invention is the Fc domain portion present in the Fc fusion proteins of any one of SEQ ID NOs 50-51, 53-54, 56-57, 59-60, as presented in Table E in section (iii) below.

[0191] In some embodiments the Fc domain(s) of a monovalent Fc fusion protein of the present invention is the Fc domain portion present in the Fc fusion proteins of any one of SEQ ID NOs 50, 53, 56, or 59, as presented in Table E in section (iii) below, wherein the sequence is an Fc “knob” chain.

[0192] In some embodiments the Fc domain(s) of a monovalent Fc fusion protein of the present invention is the Fc domain portion present in the Fc fusion proteins of any one of SEQ ID NOs 51, 54, 57, or 60, as presented in Table E in section (iii) below, wherein the sequence is an Fc “hole” chain.

[0193] IL-33 Fc Fusion Proteins

[0194] As summarized above, in one aspect the present invention provides recombinant IL-33 proteinsAtorney Docket No. MSKCC.064.WO1

[0195] Electronically filed on: January 9, 2026 that are Fc fusion proteins comprising: (a) a human IL-33 protein domain (“IL-33 domain”), as described in section (i) of the Detailed Description above, and (b) a human IgG Fc domain (“Fc domain”) as described in section (ii) of the Detailed Description above.

[0196] It is important to note that, as described above, the Fc domains and Fc fusion proteins of the present invention dimerize. The dimeric forms of the Fc fusion proteins of the present invention have two chains, which may be identical or may be different. Where the first and second chains are different they may be referred to herein as a first chain and a second chain. Thus, in some embodiments, the recombinant IL-33 proteins of the present invention are dimeric Fc fusion proteins, comprising two chains (a first chain and a second chain), wherein at least one of the two chains comprises an Fc fusion protein comprising: (a) a human IL-33 protein domain (“IL-33 domain”), as described in section (i) of the Detailed Description above, and (b) a human IgG Fc domain (“Fc domain”) as described in section (ii) of the Detailed Description above.

[0197] Further details of the Fc fusion proteins and dimeric Fc fusion proteins of the present invention are provided below.

[0198] The Fc fusion proteins or dimeric Fc fusion proteins of the present invention may comprise any of the IL-33 domains described in section (i) of the Detailed Description above linked to any of the Fc domains described in section (ii) of the Detailed Description above, in any suitable combination.

[0199] In some embodiments, the Fc fusion proteins or dimeric Fc fusion proteins comprise: (a) human IL-33 protein domain (“IL-33 domain”), and (b) a human IgGFc domain (“Fc domain”), wherein the IL-33 domain comprises one or more of the mutations described in section (i) of the Detailed Description above, and wherein the Fc domain comprises one or more mutations described in section (ii) of the Detailed Description above.

[0200] In some embodiments, the Fc fusion proteins or dimeric Fc fusion proteins comprise: (a) human IL-33 protein domain (“IL-33 domain”), and (b) a human IgGFc domain (“Fc domain”), wherein the IL-33 domain comprises any one of SEQ ID NOs. 4-30 (shown in Table B below) and wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NO. 33-65 (shown in Table F).

[0201] In some embodiments the Fc domain is located N-terminal to the IL-33 domain. In other embodiments the Fc domain is located C-terminal to the IL-33 domain.Atorney Docket No. MSKCC.064.WO1

[0202] Electronically filed on: January 9, 2026 In some embodiments the IL-33 domain is linked directly to the Fc domain by a covalent bond, with no intervening amino acids. In some embodiments the IL-33 domain is linked directly to the Fc domain by a peptide bond, with no intervening amino acids.

[0203] In some embodiments the IL-33 domain is linked to the Fc domain by a linker moiety, such as a peptide linker moiety. Any suitable linker moiety known in the art can be used. Numerous linkers suitable for use in fusion proteins are known in and can be used in the Fc fusion proteins of the present invention. For example, several suitable linkers are described in Chen et al.

[0204] “Fusion protein linkers: Property, design and functionality,” Advanced Drug Delivery Reviews, Volume 65, Issue 10, 2013, Pages 1357-1369, ISSN 0169-409X, the contents of which are hereby incorporated by reference.

[0205] In some embodiments such a linker moiety is a G / S linker composed of a stretch of 2 or more glycine and / or serine amino acids. Various different G / S linkers can be used. In some embodiments the linkers have the structure (GxS)n, where x denotes the number of G residues and n represents the number or repeats of the (GXS) unit, and wherein x and n can be from 1 upwards (e.g., from 1 to 10). For example, a (GiS)i linker has the amino acid sequence GS, a (628)2 linker has the amino acid sequence GGSGGS (SEQ ID NO. 66, a (638)3 linker has the amino acid sequence GGGSGGGSGGGS (SEQ ID NO. 67), and so on. Any (GxS)nlinker that permits activity of the IL-33 domain can be used. Accordingly, in some embodiments the linker is a (GxS)n linker. In some embodiments the linker is a (G2S)nlinker. In some embodiments the linker is a (GsS)n linker (SEQ ID NO: 68). In some embodiments the linker is a (G4S)nlinker (SEQ ID NO. 69). In some embodiments the linker is a (628)3 linker (SEQ ID NO. 70). In some embodiments the linker is a (638)3 linker (SEQ ID NO. 67). In some embodiments the linker is a (G4S)3linker (SEQ ID NO. 71).

[0206] In some embodiments, the Fc fusion proteins or dimeric Fc fusion proteins comprise any one of SEQ ID NOs. NO. 33-65 (shown in Table F).

[0207] Bivalent Fc Fusions

[0208] In some embodiments the dimeric Fc fusion proteins of the present invention are bivalent, meaning that both chains of the dimer comprise an IL-33 domain. Typically, the first and second chains of such bivalent molecules are identical to one another - i.e., they are “homodimers.” The two chains of bivalent, dimeric Fc fusion proteins can comprise any of the recombinant IL-33 proteins, IL-33 domains, Fc domains, or Fc fusion proteins described herein. Typically the FcAtorney Docket No. MSKCC.064.WO1

[0209] Electronically filed on: January 9, 2026 domains of bivalent Fc fusion proteins according to the present invention do not contain “knob” or “hole” Fc mutations.

[0210] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain (that are optionally identical), and wherein the first chain and the second chain each comprises: (a) a human IL-33 protein domain (“IL-33 domain”), as described in section (i) above, and (b) a human IgGFc domain (“Fc domain”) as described in section (ii) above.

[0211] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises any one of SEQ ID NOs. NO. 33-49, 52, 55, 58 and 61-65 (shown in Table F).

[0212] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises any one of SEQ ID NOs. NO. 38-49, 52, 55, 58 and 61-65 (shown in Table F).

[0213] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises any one of SEQ ID NOs. NO. 40-49, 52, 55, 58 and 61-65 (shown in Table F).

[0214] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises any one of SEQ ID NOs. NO. 42-49, 52, 55, 58 and 61-65 (shown in Table F).

[0215] In some embodiments, the Fc fusion proteins are bivalent dimers comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises any one of SEQ ID NOs. NO. 44-49, 52, 55, 58 and 61-65 (shown in Table F).

[0216] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 38 (shown in Table F).

[0217] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 39 (shown in Table F).

[0218] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and eachAtorney Docket No. MSKCC.064.WO1

[0219] Electronically filed on: January 9, 2026 comprises SEQ ID NO. 40 (shown in Table F).

[0220] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 41 (shown in Table F).

[0221] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 42 (shown in Table F).

[0222] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 43 (shown in Table F).

[0223] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 44 (shown in Table F).

[0224] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 45 (shown in Table F).

[0225] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 46 (shown in Table F).

[0226] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 47 (shown in Table F).

[0227] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 48 (shown in Table F).

[0228] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 49 (shown in Table F).Atorney Docket No. MSKCC.064.WO1

[0229] Electronically filed on: January 9, 2026 In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 52 (shown in Table F).

[0230] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 55 (shown in Table F).

[0231] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 58 (shown in Table F).

[0232] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 61 (shown in Table F).

[0233] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 62 (shown in Table F).

[0234] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 63 (shown in Table F).

[0235] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 64 (shown in Table F).

[0236] In some embodiments, the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and second chain are optionally identical, and each comprises SEQ ID NO. 65 (shown in Table F).

[0237] Monovalent Fc Fusions

[0238] In some embodiments the dimeric Fc fusion proteins of the present invention are monovalent, meaning that only one chain of the dimer comprises an IL-33 domain and thus the first and second chains of such monovalent molecules are not identical to one another - i.e., they are “heterodimers.” One chain of such monovalent molecules (typically referred to as the first chainAtorney Docket No. MSKCC.064.WO1

[0239] Electronically filed on: January 9, 2026 herein) can comprise any of the recombinant IL-33 proteins, IL-33 domains, Fc domains, or Fc fusion proteins described herein provided that the Fc domain comprises a “knob” or “hole” mutation(s). The other chain of such monovalent molecules (typically referred to as the second chain herein) comprises an Fc domain but does not comprise an IL-33 domain or an Fc fusion protein. The Fc domain in this chain (the second chain) can comprise any of the Fc domains described herein provided that the Fc domain comprises a “knob” or “hole” mutation(s). If one chain (the first chain) comprises a “knob” mutation the other chain (the second chain) should comprise “hole” mutations. Conversely, if one chain (the first chain) comprises “hole” mutations the other chain (second chain) should comprise a “knob” mutation. In some embodiments the chain that comprises the IL-33 domain is the knob chain and the chain that does not comprise IL-33 in the hole chain. In other embodiments the chain that comprises the IL-33 domain is the hole chain and the chain that does not comprise IL-33 in the knob chain. While the former configuration (where the chain that comprises the IL-33 domain is the knob chain) is preferred, either configuration may be used.

[0240] In some embodiments, the Fc fusion proteins are monovalent dimers comprising a first chain and a second chain, wherein the first chain comprises: (a) a human IL-33 protein domain (“IL-33 domain”), as described in section (i) above, and (b) a human IgGFc domain (“Fc domain”) as described in section (ii) above, and the second chain comprises a human IgGFc domain (“Fc domain”) as described in section (ii) above and does not comprise an IL-33 domain.

[0241] In some embodiments, the Fc fusion proteins are monovalent dimers comprising a first chain and a second chain, where the first chain comprises any one of SEQ ID NOs. 50, 53, 56, or 59 and the second chain comprises any one of SEQ ID NOs. 51, 54, 57, 60 (shown in Table F).

[0242] In some embodiments, the Fc fusion protein is a monovalent dimer comprising a first chain and a second chain, where the first chain comprises SEQ ID NO. 50, and the second chain comprises SEQ ID NO. 51. (shown in Table F). This molecule is referred to herein as Mo_Fcl_M14-IL-33.

[0243] In some embodiments, the Fc fusion protein is a monovalent dimer comprising a first chain and a second chain, where the first chain comprises SEQ ID NO. 53, and the second chain comprises SEQ ID NO. 54. (shown in Table F). This molecule is referred to herein as Mo_aFcl_M 14 -IL-33.

[0244] In some embodiments, the Fc fusion protein is a monovalent dimer comprising a first chain and a second chain, where the first chain comprises SEQ ID NO. 56, and the second chain comprisesAttorney Docket No. MSKCC.064.WO1

[0245] Electronically filed on: January 9, 2026 SEQ ID NO. 57. (shown in Table F). This molecule is referred to herein as Mo_aFc_M14-IL-33.

[0246] In some embodiments, the Fc fusion protein is a monovalent dimer comprising a first chain and a second chain, where the first chain comprises SEQ ID NO. 59, and the second chain comprises SEQ ID NO. 60. (shown in Table F). This molecule is referred to herein as Mo_Fcl_M15-IL-33.

[0247] Table F: Amino Acid Sequences of IL-33 - Fc Fusion Proteins

[0248]

[0249] Atorney Docket No. MSKCC.064.WO1

[0250] Electronically filed on: January 9, 2026

[0251]

[0252] Atorney Docket No. MSKCC.064.WO1

[0253] Electronically filed on: January 9, 2026

[0254]

[0255] Atorney Docket No. MSKCC.064.WO1

[0256] Electronically filed on: January 9, 2026

[0257]

[0258] Atorney Docket No. MSKCC.064.WO1

[0259] Electronically filed on: January 9, 2026

[0260]

[0261] Atorney Docket No. MSKCC.064.WO1

[0262] Electronically filed on: January 9, 2026

[0263]

[0264] Atorney Docket No. MSKCC.064.WO1

[0265] Electronically filed on: January 9, 2026

[0266]

[0267] In Table F, above, the bold residues are the Fc domain sequence, the underlined residues are the IL-33 domain sequence, and the intervening residues are the linker sequence. The fusion proteins listed in Table F were named as a composite of the constituent IL-33 and Fc components (defined in Tables C and E), with fusion orientation defined by the ordering of “Fc” and “IL-33” within a given construct name. For example, IL-33_Bi_Fc0 and Bi_Fc0_IL-33 are fusions of wild type IL-33 to the N- and C-terminus, respectively, of IgGl (L234A / L235A) Fc.

[0268] Methods of Manufacture

[0269] The recombinant IL-33 proteins described herein can be made using any suitable method known in the art for the production of recombinant proteins, including, but not limited to, by expression from a nucleic acid molecule encoding a recombinant IL-33 proteins as described herein, and by chemical synthesis, for example, using solid-phase peptide synthesis or solution-method peptide synthesis.

[0270] The recombinant IL-33 proteins of the present invention can be produced by expression from a nucleic acid molecule in any suitable host cell type, including, but not limited to eukaryotic cells, prokaryotic cells, bacteria, mammalian cells, (such as Chinese Hamster Ovary or “CHO” cells), and insect cells. In addition, cell free recombinant protein expression (also known as in vitro expression) may be used to produce the recombinant IL-33 proteins of the present invention. Methods for expression of recombinant proteins from nucleic acid molecules that encode them are routine and well known in the art, and any suitable methods, vectors, systems, and / or host cell types known in the art can be used. For example, nucleic acid molecules encoding the recombinant IL-33 proteins described herein can be produced (e.g., by cloning or by chemical synthesis using an oligonucleotide synthesizer, cloned into a suitable expression construct containing a suitable promoter to drive expression, and delivered to host cells (e.g., by transduction or transfection), such that the recombinant IL-33 proteins encoded by the nucleic acid molecules will be expressed by the host cells (by transcription and translation) and can thenAtorney Docket No. MSKCC.064.WO1

[0271] Electronically filed on: January 9, 2026 be purified therefrom. The nucleotide sequences of nucleic acid molecules encoding the recombinant IL-33 proteins of the invention can readily be deduced using the genetic code and selecting a sequence of codons that encode each amino acid in the amino acid sequence of the recombinant IL-33 protein. In some embodiments, the codons used may be selected to be those favored in the host cell in which the recombinant IL-33 protein will be expressed.

[0272] The recombinant IL-33 proteins of the present invention can be produced by chemical synthesis. Methods for chemically synthesizing proteins are routine and well known in the art. Proteins can be synthesized from amino acids (including non-canonical amino acids and / or modified amino acids) and amino acid analogues that are themselves either synthesized using standard methods known in the art or are obtained from commercial sources. Methods for chemically synthesizing peptides using solid-phase peptide synthesis or solution-method peptide synthesis are routine and well known in the art.

[0273] As described above, many of the recombinant IL-33 proteins of the present invention are, or form, dimers comprising a first and second chain. The dimers will form spontaneously when the first and second chain are in close proximity to one another, for example, after expression (e.g., co-expression) of a nucleotide sequence encoding the first chain and a nucleotide sequence encoding the second chain. When the dimers are bivalent, production of the dimer is entirely homodimeric. When the dimers are monovalent, with only one chain having an IL-33 domain, the monovalent dimers are the preferred heterodimeric species but contaminating homodimers may also be produced. The use of mutations in chains 1 and 2 (such as the knob-in-hole mutations described above) promotes formation of the desired heterodimeric species in excess over undesired homodimer contaminants, and additional purification steps may be employed if desired to select the desired heterodimeric species.

[0274] In some embodiments, the recombinant IL-33 proteins of the present invention can be produced as isolated or purified proteins. In such embodiments the recombinant IL-33 proteins may be purified away from certain other components, such as certain other components that are used in or result from the method used to manufacture the recombinant IL-33 proteins such as host cells, host cell proteins, solvents, and / or any other chemicals or other agents or cellular components used in or produced as part of the manufacturing process. The recombinant IL-33 proteins may be purified away from such other components using any suitable method known in the art including, but are not limited to, chromatography (e.g., ion exchange, affinity, and / or sizing column chromatography), ammonium sulfate precipitation, centrifugation, differential solubility,Atorney Docket No. MSKCC.064.WO1

[0275] Electronically filed on: January 9, 2026 or by any other technique for the purification of proteins known to one of ordinary skill in the art. In some embodiments the recombinant IL-33 proteins are purified by Protein A affinity chromatography. In some embodiments, the isolated or purified proteins are provided in a form in which they comprise more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5%, or 99.9% of the total protein content of the final composition.

[0276] In some embodiments, the recombinant IL-33 proteins of the present invention are analyzed following manufacture to confirm successful production (e.g., successful dimerization), for example using mass spectrometry (MS), liquid chromatography (e.g., HPLC), NMR (e.g., high-resolution multi-dimensional NMR), immunobloting, functional assays, or any other suitable analysis method known in the art.

[0277] In some embodiments, the recombinant IL-33 proteins of the present invention are analyzed following manufacture to confirm that they have the desired activity. Various methods of assessing such activity are known in the art, including those described in the Examples section of this patent disclosure.

[0278] Nucleic Acid Molecules, Vectors, and Host Cells

[0279] In some embodiments, the present invention provides nucleic acid molecules that encode the recombinant IL-33 proteins of the present invention. Similarly, in some embodiments, the present invention provides vectors that comprise such nucleic acid molecules. Similarly, in some embodiments, the present invention also provides host cells that comprise such nucleic acid molecules or vectors - for example host cells that have been engineered to produce one or more of the recombinant IL-33 proteins of the present invention by recombinant expression.

[0280] One of ordinary skill in the art can readily determine the nucleotide sequence of a nucleic acid molecule that encodes any one of the recombinant IL-33 proteins described herein - given the universally known and understood nature of the genetic code. Nucleic acid molecules encoding the recombinant IL-33 proteins of the invention can be designed by identifying the codons in the genetic code that encode each amino acid in the desired recombinant IL-33 protein and, optionally, by selecting those codons that are favored in the host cell in which the peptide will be expressed. In this manner a nucleotide sequence encoding a recombinant IL-33 protein of the present invention can be determined and a nucleic acid molecule having that nucleotide sequence can be produced using standard techniques known in the art, such as by cloning or by using anAtorney Docket No. MSKCC.064.WO1

[0281] Electronically filed on: January 9, 2026 oligonucleotide synthesizer.

[0282] In some embodiments, a nucleic acid molecule encoding a recombinant IL-33 protein of the present invention can be cloned into any suitable vector, such as those to be used for propagation of the nucleic acid molecule or those to be used for expression (i.e., an expression vector) - i.e., to produce the recombinant IL-33 protein encoded by the nucleic acid molecule by a process of transcription and translation. In embodiments requiring expression, the nucleic acid can be operatively linked to a promoter suitable for directing expression in the desired host cell type, such as a eukaryotic or prokaryotic cell, and may be incorporated into any suitable expression vector.

[0283] In some embodiments, nucleic acid molecules encoding the recombinant IL-33 proteins of the present invention can be codon optimized for expression in cells of a particular organism or species.

[0284] Compositions

[0285] In some embodiments, the present invention provides compositions comprising one or more of the recombinant IL-33 proteins described herein and one or more additional components. In some embodiments, such compositions may be pharmaceutical compositions.

[0286] As used herein, the term “pharmaceutical composition” refers to a composition the components of which permit the biological activity of the active agent(s) (e.g., proteins) contained therein, and that contain no additional components that are unacceptably toxic to a living subject to which the composition may be administered. Thus, the additional components included in pharmaceutical compositions according to the present invention will typically be components known to be safe for administration to human and / or other animal subjects, and / or approved by a regulatory agency of the Federal or a state government, and / or listed in the U.S. Pharmacopeia, and / or other generally recognized pharmacopeia, and / or receiving specific or individual approval from one or more generally recognized regulatory agencies for use in humans and / or other animals. Typically, a pharmaceutical composition according to the present invention will contain an effective amount of a recombinant IL-33 protein of the invention, preferably in a purified form, together with a suitable amount of one or more pharmaceutically acceptable carriers and / or excipients.

[0287] Suitable pharmaceutically acceptable carriers and / or excipients include various diluents andAtorney Docket No. MSKCC.064.WO1

[0288] Electronically filed on: January 9, 2026 vehicles in which, or with which, the recombinant IL-33 proteins of the present invention can be provided, including, but not limited to, water, aqueous solutions (such as saline solutions, aqueous dextrose solutions, buffers, and the like), organic solvents (such as polyethylene glycols, glycerin, propylene glycol, certain alcohols (such as ethanol) and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil), glycerol, and the like.

[0289] Additional carriers and / or excipients that can be used include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, weting agents, emulsifying agents, pH buffering agents, antimicrobial agents, preservatives, and the like.

[0290] In some embodiments, the present invention provides compositions (e.g., pharmaceutical compositions) that comprise a recombinant IL-33 protein of the invention in water. In some embodiments, the present invention provides compositions (e.g., pharmaceutical compositions) that comprise a recombinant IL-33 protein of the invention in saline. In some embodiments, the present invention provides compositions (e.g., pharmaceutical compositions) that comprise a recombinant IL-33 protein of the invention in lyophilized form.

[0291] In some embodiments, the recombinant IL-33 proteins described herein may be provided in a composition (e.g., pharmaceutical composition) that comprises one or more “additional active agents” (i.e., in addition to the recombinant IL-33 proteins of the present invention and additional components described above).

[0292] In some embodiments, the present invention provides compositions (e.g., pharmaceutical compositions) that comprise an effective amount of a recombinant IL-33 protein of the invention.

[0293] In some embodiments, the compositions (e.g., pharmaceutical compositions) of the present invention are provided in a dosage form. Such forms may be solutions, suspensions, emulsions, tablets, pills, suppositories, capsules, lozenges, wafers, powders (e.g. lyophilized powders), gels, creams, ointments, foams, pastes, patches, ampoules, vials, pre-filled syringes, sustained-release formulations, and the like.

[0294] In some embodiments, the compositions (e.g., pharmaceutical compositions) of the invention may be conveniently provided in unit dosage forms. Unit dosage forms are those containing aAtorney Docket No. MSKCC.064.WO1

[0295] Electronically filed on: January 9, 2026 single dose or unit (e.g., an effective amount), or an appropriate fraction thereof, of the recombinant IL-33 proteins of the invention in of the invention. The unit dosage forms may be presented in single-dose or multi-dose containers.

[0296] In some embodiments, the compositions (e.g., pharmaceutical compositions) of the invention may be provided in sealed ampoules or vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier (e.g., water) immediately prior to use.

[0297] Kits

[0298] To facilitate use of the invention, any of the recombinant IL-33 proteins and / or compositions described herein can be provided in the form of a kit. Such a kit will typically comprise one or more recombinant IL-33 proteins and / or compositions described herein together with one or more of a container, a delivery device, instructions for use (e.g., instructions regarding the timing and / or route of administration, instructions regarding dosing, etc.) packaging materials, and the like.

[0299] Methods of Use and Methods of Treatment

[0300] The present invention provides various methods of use of the recombinant IL-33 proteins and compositions of the present invention.

[0301] For example, in some embodiments, the present invention provides methods of activating IL-33 receptors in vivo or in vitro, such methods comprising contacting an IL-33 receptor, or a cell comprising a IL-33 receptor, with a recombinant IL-33 protein or composition of the present invention.

[0302] In some embodiments, the present invention provides methods of expanding or activating ILC2 cells in vivo or in vitro / ex vivo, such methods comprising contacting ILC2 cells with a recombinant IL-33 protein or composition of the present invention.

[0303] Similarly, in some embodiments, the present invention provides methods of treating subjects having a condition amenable to treatment with IL-33 or an IL-33 receptor agonist, such methods comprising administering an effective amount of a recombinant IL-33 protein or pharmaceutical composition of the present invention to such subjects.

[0304] In some embodiments, the present invention provides methods of treating cancer in subjects inAtorney Docket No. MSKCC.064.WO1

[0305] Electronically filed on: January 9, 2026 need thereof, such methods comprising administering an effective amount of a recombinant IL-33 protein or pharmaceutical composition of the present invention to the subject. In some such embodiments the cancer is pancreatic cancer. In some such embodiments the cancer is pancreatic ductal adenocarcinoma (PDAC). In some such embodiments the cancer is an immune checkpoint inhibitor resistant cancer (such as an immune checkpoint inhibitor resistant PDAC). In some such embodiments the cancer is a PD-1 / PD-L1 inhibitor resistant cancer (such as an immune checkpoint inhibitor resistant PDAC).

[0306] Subjects that can be treated using the methods and compositions of the present invention include any mammalian subjects in which the recombinant IL-33 proteins of the invention can bind to and / or activate IL-33 receptors. In some embodiments, the subjects are non-human mammals useful for conducting preclinical studies, such as rodents (e.g., mice or rats) or non-human primates. In some embodiments, the subjects are humans.

[0307] The treatment methods provided herein involve administering the recombinant IL-33 proteins or pharmaceutical compositions of the present invention to subjects. Various routes and systems for administration are known in the art and any such suitable route or system can be used to administer the of a recombinant IL-33 protein and pharmaceutical compositions of the present invention to subjects. In some embodiments, parenteral administration is used. In some embodiments, enteral administration is used. Contemplated parenteral or enteral administration routes include, but are not limited to, intradermal, intramuscular (IM), intraperitoneal (IP), intravenous (IV) (e.g., as a bolus injection or by infusion), subcutaneous, intranasal, epidural, intracerebral, intrathecal, topical, transepithelial (e.g., by absorption through oral, rectal, gastric, esophageal, or intestinal mucosa, etc.), sublingual, and oral (PO) administration routes. In some embodiments, administration is systemic. In some embodiments, administration is local. In some embodiments, administration is intravenous (IV). In some embodiments, administration is subcutaneous (SC).

[0308] In some embodiments, an “effective amount” of the recombinant IL-33 proteins or compositions of the present invention is administered to subjects. The “effective amount” in a given situation may depend on variety of factors including, but not limited to, the species of the subject (e.g., whether a human or some other animal species), the age of the subject, the sex of the subject, the weight of the subject, the formulation of the of a recombinant IL-33 protein, the route of administration, the planned dosing regimen, etc. The effective amount (which may be a range of effective amounts) can be determined by standard techniques, for example using in vitro assaysAtorney Docket No. MSKCC.064.WO1

[0309] Electronically filed on: January 9, 2026 and / or in vivo assays in the intended subject species or any suitable animal model species.

[0310] Suitable methods for determining an effective amount include, but are not limited to, doseescalation studies, extrapolation from dose-response curves, and / or extrapolation from other data derived from in vitro and / or in vivo model systems. In some embodiments, the effective amount may be determined according to the judgment of a medical or veterinary practitioner based on the specific circumstances.

[0311] In some embodiments administration of the recombinant IL-33 proteins or pharmaceutical compositions of the present invention can be performed in conjunction with other treatment methods known to be useful for tumor therapy, including, but not limited to, surgical methods (e.g. for tumor resection), radiation therapy methods, treatment with chemotherapeutic agents, treatment with antiangiogenic agents, treatment with tyrosine kinase inhibitors or treatment with immune checkpoint inhibitors. Similarly, in certain embodiments the methods of treatment provided herein may be employed together with procedures used to monitor disease status / progression, such as biopsy methods and diagnostic methods (e.g. MRI methods or other imaging methods).

[0312] In some embodiments, administration of the recombinant IL-33 proteins or pharmaceutical compositions of the present invention can be performed in conjunction with administration of one or more additional active agents. Examples of such additional active agents include, but are not limited to immune checkpoint inhibitors, such as PD-1 and / or PD-L1 inhibitors.

[0313] In further embodiments the present invention also provides methods of treatment that comprise administering to a subject in need thereof an effective amount of a cell or vector or nucleic acid sequence capable of causing the expression and / or production of one or more of the recombinant IL-33 proteins of the present invention within the subject.

[0314] Those of skill in the art will appreciate that, in addition to being useful in methods of treatment, the recombinant IL-33 proteins of the invention may be useful for a variety of other applications. For example, the recombinant IL-33 proteins of the invention may be useful as research tools (e.g., for studying the effects of IL-33 receptor activation in vitro or in vivo), and / or as diagnostic tools (e.g., for detecting, assaying, and / or quantifying IL-33 receptors or IL-33 activity). All such uses are within the scope of the present invention.

[0315] The invention is further described in the following non-limiting Examples.Atorney Docket No. MSKCC.064.WO1

[0316] Electronically filed on: January 9, 2026 EXAMPLES

[0317] Interleukin 33 (IL-33) is an IL-1 family alarmin cytokine that is proinflammatory and functions as an endogenous danger signal. It is constitutively expressed and localizes into the nucleus [1], Upon tissue damage, full-length human IL-33 (30kDa, 270 amino acids) is released and processed by inflammatory proteases into a shorter mature form (18kDa, 159 amino acids corresponding to amino acidsl 12-270 of the mature form). Mature IL-33 contains the IL-1 like cytokine domain and is 10-30 fold more potent than the full-length form [2, 3], Mature IL-33 activates immune cells by binding and signaling through a heterodimer receptor constituted by the primary receptor IL-1 receptor like 1 - also referred to as ST2. The IL-33 / ST2 complex then recruits the co-receptor IL-1 receptor accessory protein (IL-lRAcP) assembling in a heterotrimeric complex [4, 5],

[0318] While IL-33 ’s proinflammatory function has been well known for some time, more recently it has been discovered that IL-33 may also play a role in anti-cancer immunity through activation of immune cells such as dendritic cells (DCs) [9], cytotoxic anti-cancer effector T cells [10, 11], natural killer cells (NK cells)

[0012] , eosinophils [13, 14], and, importantly, group 2 innate lymphoid cells (ILC2s)

[0015] , Indeed, it appears that wild-type IL-33 can drive anti-tumor immunity either alone or in combination with checkpoint blockade agents

[0015] ,

[0319] However, there are several barriers to using wild-type IL-33 as a cancer therapeutic. For example, to limit the potent proinflammatory effects of IL-33 alarmin, several natural mechanisms exist that reduce IL-33 bioactivity in vivo. These include: (1) rapid inactivation in the extracellular environment by oxidation of 4 cysteines leading to the formation of two disulfide bridges, which disrupt binding to ST2 due to a conformational change [6]; (2) IL-33 neutralization by decoy receptors such as soluble ST2 and IL-lRacP [7, 8]; and (3) proteolytic processing [1, 2], In addition, the small size of the mature wild-type IL-33 molecule results in it being rapidly cleared from the circulation. The wild-type / native form of IL-33 therefore has limited utility as a therapeutic agent.

[0320] To overcome these significant obstacles, we aimed to engineer an improved form of IL-33 that has a longer half-life and exhibits resistance to cysteine oxidation and proteolysis - making it potentially more useful as a therapeutic agent. We employed extensive protein engineering efforts to address each of the liabilities of wild-type IL-33 described above, resulting in the creation of several novel recombinant IL-33 proteins (including disulfide-stabilized IL-33 Fc-Atorney Docket No. MSKCC.064.WO1

[0321] Electronically filed on: January 9, 2026 fusion proteins). Importantly, in addition to addressing the therapeutic liabilities associated with wild-type IL-33 described above, our novel recombinant IL-33 proteins also exhibit robust activation of IL-33 signaling and treatment efficacy in preclinical pancreatic cancer models. The following Examples describe the work performed to engineer the IL-33 molecule, the novel recombinant IL-33 proteins that were produced, and the testing and biological activity of these novel recombinant IL-33 proteins in vitro and in vivo.

[0322] EXAMPLE 1

[0323] Production of Initial Engineered IL-33 Fc Fusion Proteins

[0324] To increase IL-33 ’s half-life and improve protein production, a number of recombinant IL-33 proteins were designed and tested. These proteins were Fc fusion proteins - comprising fusions of mature wild-type human IL-33 (amino acids 112-270) or mutant forms thereof to human IgGl Fc or human IgG4 Fc in two orientations (the Fc was fused to either the N- or C-terminus of IL-33 via a 12 residue Gly / Ser-containing linker). The IgGl Fc fragment consisted of amino acids 221-447 (Eu numbering) while the IgG4 Fc fragment consisted of amino acids 216-447. In addition, a monovalent IL-33 Fc fusion was generated employing knob-in-hole (KiH) Fc mutations. Validated mutations in the Fc region were also used to silence Fc / antibody effector function. The design of these recombinant IL-33 proteins is illustrated schematically in Fig. 1.

[0325] The 5 recombinant IL-33 protein constructs shown in Fig. 1 are named according to the schema defined in Table F in the Detailed Description.

[0326] Purified fusion proteins were assayed for IL-33 activity using a commercially available HEK-Blue™ reporter assay from InvivoGen in which HEK-Blue™ IL-33 reporter cells are stably transfected to express human ST2 and an NF-KB / AP-1 -inducible secreted embryonic alkaline phosphatase (SEAP) reporter. The other IL-33 receptor subunit, IL-lRAcP, is naturally expressed in these cells, so when stimulated with IL-33, pathway activation leads to induced SEAP expression which can be quantitated in culture supernatants using QUANTLBlue™ Solution detection reagent. Although the 5 fusion proteins shown in Fig. 1 were active in the reporter assay, they were significantly less active than wild type IL-33. Moreover, test samples contained significant levels of aggregate upon Protein A (Pro- A) affinity purification, as determined by analytical size exclusion chromatography (SEC). This is exemplified by the IgG4 Fc fusion constructs Bi_Fc4-IL-33 and IL-33_Bi_Fc4 (Table 1). We hypothesized that this extensive aggregation and poor bioactivity were likely a result of the unpaired cysteine residuesAttorney Docket No. MSKCC.064.WO1

[0327] Electronically filed on: January 9, 2026 which had not been modified in these initial constructs (See Table F in the Detailed Description).

[0328] To address the unpaired cysteine liabilities, four substitutions were introduced to replace these residues with serine (C208S, C227S, C232S and C259S). The resultant IL-33 variant (CS-IL-33;

[0329] Appendix A, Table SI) was fused C-terminally to effector-silenced human IgGl or IgG4 Fc regions and both monovalent and bivalent Fc fusions were generated. Both mono and bivalent IL-33-CS Fc-fusions exhibited considerably less aggregation and were significantly more bioactive in the HEK-Blue™ reporter assay (Table 1). As no major differences in yield, purity or bioactivity were observed between the IgGl and IgG4 Fc fusions, the hlgGl “LALA" mutation fusion partner (Bi FcO; Table F in the Detailed Description) was selected for further development.

[0330] Table 1: Productivity attributes and bioactivity of IL-33 and CS-IL-33 Fc-fusion proteins

[0331] < > < >

[0332] > >

[0333] > >

[0334] > >

[0335]

[0336] * Yield per L culture supernatant after single column Pro-A purification

[0337] AYield per L culture supernatant after additional SEC purification

[0338] EXAMPLE 2

[0339] Identification and Removal of IL-33 Glycosylation Liability Site

[0340] As an intracellular protein, wild-type IL-33 is not naturally glycosylated but the sequence contains an N-X-S / T motif at amino acid residues 171-173 (NES) which could potentially be N-Atorney Docket No. MSKCC.064.WO1

[0341] Electronically filed on: January 9, 2026 glycosylated when recombinant protein is produced from an eukaryotic expression host as a secreted product. The C259S mutation described above (present in CS-IL-33) also introduces a second N-X-S / T motif and possible glycosylation site at amino acid residues 257-259 (NLS). Glycosylation at either site would be unnatural and could potentially lead to product heterogeneity and negatively impact pharmacokinetics due to uptake by asialoglycoprotein receptors. Therefore, the glycosylation status of the candidate fusion molecules was investigated. Intact mass analysis of recombinant Bi_Fc0_CS-IL-33 fusion protein produced in CHO-express cells was performed as follows. Electrospray ionization liquid chromatography mass spectrometry (ESI / LC / MS) was used to confirm molecular weight (MW) of target protein fusion, with and free of glycans, and assess heterogeneity of the fusion protein. The Fc-fusion protein samples were reduced under a variety of conditions ranging from 20 mM to 50 mM dithiothreitol (DTT) at pH 8.6 for 15 min to 1 hr at 60-80°C. HPLC column: 2.1x50 mm Halo Diphenyl 2.7 pl. HPLC conditions for reduced Fc-fusion protein, flow rate: 500 pl / min, mobile phase: Solvent A = 0.1% aqueous TFA; Solvent B = ACN / 0.1% aqueous TFA. Initial conditions: 10% B then 20% B for 1 min, 20-50% B gradient for 8 min and 70% B for 2 min. Respective ESEMS spectra were deconvoluted using ProMass software.

[0342] It was observed by mass spectrometry (MS) that the fusion protein was glycosylated. To determine whether the N171 residue at site 1 (NES) and / or the N257 residue at site 2 (NLS) were glycosylated, mutations were introduced into IL-33 at site 1 (NES) and at site 2 (NLS). First, an S173A mutation was introduced to eliminate the N-X-S motif at site 1. Analysis of this material by MS indicated that the S173A variant of Bi_Fc0_CS-IL-33 (Bi_Fc0_ACS-IL-33;Table F in the Detailed Description ) lacked a higher mass glycosylated species that was detected in Bi_Fc0_CS-IL-33, confirming occupancy of site 1 by glycan in this molecule (Fig.2 and Table 2).

[0343] Table 2: Identification of N-linked Glycosylation in IL-33 Region of Fc-IL-33

[0344]

[0345] Atorney Docket No. MSKCC.064.WO1

[0346] Electronically filed on: January 9, 2026

[0347]

[0348] Although our data did not reveal glycosylation of site 2 in Bi_Fc0_ACS-IL-33, to eliminate the possibility of glycosylation at this N-X-S motif (which, as described above, was introduced as a consequence of the C259S substitution), an alternative substitution (C259A) was introduced in Bi_Fc0_ACS-IL-33 (Bi_Fc0_M0-IL-33, Table F in the Detailed Description) to both eliminate the unpaired cysteine liability and also remove the N-X-S potential glycosylation motif. As expected, MS analysis of Bi_Fc0_M0-IL-33 was consistent with glycosylation solely within the Fc region atN297. We also re-evaluated the use of serine substitutions for other unpaired cysteines and determined that the C227S substitution described above had introduced a potential deamidation motif (NS) at position 226. Accordingly, an alternative substitution (C227A instead of C227S) was incorporated into Bi_Fc0_M0-IL-33 background (Bi_Fcl_Ml-IL-33; Table F in the Detailed Description). Importantly, both of these variants possessed equivalent bioactivity to IL-33 glycosylated Bi_Fc0_CS-IL-33 (Fig. 14; Table F), thus, Bi_Fcl_Ml-IL-33 was selected for further optimization.

[0349] EXAMPLE 3

[0350] IL-33 Fc Fusion Proteins are Prone to Proteolytic Degradation

[0351] Although Bi_Fcl_Ml-IL-33 had favorable expression yields and lacked glycosylation and oxidation liabilities within IL-33, it was noted that material produced transiently in CHO-express™ cells (GenScript) and purified by Pro-A affinity chromatography exhibited significant fragmentation (>25%) within the purified product as observed by MS analysis (Fig. 15). The mass of the main fragmentation product was consistent with cleavage within a long unstructured loop of IL-33 (aa 165 - 182) that is a known site for proteolytic inactivation by cellular proteases. Additional fragmentation was further observed when purified material was incubated in human serum, suggesting overall susceptibility to proteolysis (Fig. 16). Given that the production and serum stability issues identified pose a significant challenge to pre-clinical development, further engineering of the fusion protein was warranted.

[0352] EXAMPLE 4

[0353] Design of Disulfide-Containing IL-33 Fc Fusion VariantsAtorney Docket No. MSKCC.064.WO1

[0354] Electronically filed on: January 9, 2026 We hypothesized that it might be possible to stabilize our IL-33 fusion variants by introducing disulfide bonds. The introduction of disulfide bonds must be carefully undertaken to avoid distortion to the protein structure. Therefore, potential disulfide bonds were designed using two available crystal structures of IL-33 (the crystal structure of human IL-33 complexed with ST2 (PDB: 4KC3), and the crystal structure of the ternary complex of mouse IL-33 bound to ST2 and IL-lRAcP (PDB: 5VI4). Briefly, in silico engineering using Schrodinger software BioLuminate (cysteine mutation tool) and available crystal structures of hIL-33 complexed with ST2 (PDB: 4KC3) and tertiary complex mIL-33 / ST2 / ILlRacP (PDB: 5VI4), disulfides were introduced in IL-33 [Salam.NK, Prot. Eng. Design & Selection, 2014], The following design criteria were employed: 1) Distance criteria (< 6.5 Angstrom between the two C-beta); 2) low BioLuminate computational energy score and 3) avoid position involved in ST2 / ILlRacP receptor interface. Using a 6.5 A CP-CP cut-off distance, several potential disulfide bonds that could be formed by substitution of IL-33 residues or residue pairs with cysteine were identified.

[0355] A weighted score was computed for each of the designed disulfide bonds such that lower scores were ranked most favorably. After we excluded IL-33 disulfide variants with cysteine mutations in the ST2 / IL-lRAcP receptor interface, we selected 24 variants for experimental evaluation by screening for binding to ST2 receptor and functional activity in a reporter cell assay (variants M2-through M25-IL-33, Table C in the Detailed Description and Table 3.

[0356] Table 3: Design of Di sulfide- Stabilized IL-33 Variants

[0357]

[0358] Atorney Docket No. MSKCC.064.WO1

[0359] Electronically filed on: January 9, 2026

[0360]

[0361] In Table 3 “CA” is the wild type cysteine residue at amino acid position 232.

[0362] The 24 candidates, along with the parent IL-33 scaffold (Ml -IL-33), were cloned as fusions to a C-terminal glycosylphosphatidylinositol (GPI) anchor to enable cell-surface display

[0017] , Constructs also contained an N-terminal Myc tag to allow for assessment of relative expression levels. Following transient transfection of 293 cells with these constructs, flow cytometry was used to assess surface display levels (Fig.3A) and binding to soluble ST2 protein (Fig.3B).

[0363] While expression of all 24 constructs was similar based on Myc-tag staining, the low sensitivity of the ST2 binding assay precluded definitive assessment of ST2 binding capacity for the entire panel of variants. However, 12 of the 24 cell-surface displayed IL-33 variants exhibited ST2 binding signals that were greater than that of the Ml -IL-33 control. Thus, these candidates were prioritized for further evaluation. Expi-CHO cells were transiently transfected with this subset of GPLanchored variants, then treated with phosphatidylinositol-specific phospholipase C to release soluble proteins from the anchor. Supernatants from the lipase-treated cultures were then evaluated in the IL-33 reporter assay (Fig. 3C). All 12 of these IL-33 disulfide variants were bioactive in the IL-33 reporter assay.

[0364] EXAMPLE 5

[0365] Production, Analytical and Stability Characterization of Disulfide Containing IL-33 Fc FusionAtorney Docket No. MSKCC.064.WO1

[0366] Electronically filed on: January 9, 2026 Variants

[0367] To further assess production, stability and functional atributes for the 12 bioactive IL-33 variants, the 12 variants were formatted as bivalent Fc (Bi Fcl) fusions (Table F), expressed recombinantly in transiently transfected expi-CHO cells, and purified by standard Pro-A affinity chromatography. Following purification, production attributes of fragmentation and aggregation were assessed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. 4) and analytical size exclusion chromatography (SEC) (Fig. 5). The majority of the disulfide-containing IL-33 Fc fusion variants demonstrated reduced levels of protein fragmentation, as assessed by SDS-PAGE, compared to the non-disulfide containing Bi_Fcl_Ml-IL-33. In particular, variants Bi_Fcl_M3-IL-33, Bi_Fcl_M14-IL-33, Bi_Fcl_M15-IL-33 and Bi_Fcl_M16-IL-33 exhibited the lowest levels of degradation (Fig. 4).

[0368] Consistent with the SDS-PAGE data, differing levels of product fragmentation was also apparent in the analytical SEC profiles for the 12 disulfide-stabilized fusion variants (Fig. 5). Main peak tailing was apparent for Bi_Fcl_Ml-IL-33 and a number of other variants, indicative of lower molecular weight fragmentation products. A number of samples exhibited better main peak symmetry, which generally correlated with higher purity as assessed by SDS-PAGE. In particular, main peak symmetry for M3-, M14-, M15- and M16-containing fusions was greater than the nonstabilized Bi_Fcl_Ml-IL-33 reference (Fig. 5), reflecting their enhanced stability and reduction in susceptibility to proteolytic degradation.

[0369] EXAMPLE 6

[0370] Functional Characterization of Disulfide-Containing IL-33-Fc Fusion Proteins

[0371] To assess the 12 disulfide-stabilized fusion variants for functional activity, purified samples were evaluated in the HEK-Blue™ IL-33 reporter assay (Fig. 6). All variants were active in this assay and relative differences in potency were modest with a 6-fold difference between the highest and lowest potency samples based on concentrations required to achieve a half-maximal response (highest potency: Bi_Fcl_M12-IL-33; EC 50 = 0.09 nM, lowest: Bi_Fcl_M25-IL-33; EC50 = 0.50 nM). The M3-, M14-, M15- and Ml 6-IL-33 -containing variants that exhibited the best productivity and purity data were also favorable with respect to potency in the reporter cell assay, with EC50 values that were modestly improved (0.11 - 0.15 nM) relative to that of the nonstabilized Bi_Fcl_Ml-IL-33 reference (0.24 nM).Atorney Docket No. MSKCC.064.WO1

[0372] Electronically filed on: January 9, 2026 EXAMPLE 7

[0373] Di sulfide- Stabilized IL-33 Fc Fusion Proteins Exhibit Enhanced Resistance to Proteolysis and Serum Stability

[0374] To further assess whether M3-, M14-, M15- and Ml 6-IL-33 -containing fusion variants had enhanced resistance to proteolysis, they were incubated with the broad-specificity protease thermolysin and the kinetics of degradation were compared to that of the non-stabilized Bi_Fcl_Ml-IL-33 reference. Briefly, IL-33-Fc variants were diluted to 0.5 mg / mL in PBS buffer containing 0.2mM CaCh and equilibrated to 37°C. A small volume of thermolysin solution from a freshly prepared stock was added to initiate proteolysis, such that the final substratethermolysin ratio was 50:1 on a mass basis. Proteolysis was stopped at the indicated times by addition of 2 mM EDTA which inactivates thermolysin through sequestration of catalytic zinc and structural calcium ions. Inactivated samples were stored at 4°C prior to analysis by reducing SDS-PAGE. While thermolysin rapidly cleaved the linkage between the Fc and IL-33 region for all constructs, there were significant differences in the subsequent rate of degradation of the liberated IL-33 fragment. All 4 of the disulfide-containing variants exhibited superior resistance to degradation relative to the non-stabilized Bi_Fcl_Ml-IL-33 molecule, with Bi_Fcl_M14-IL-33 showing the slowest rate of IL-33 degradation. While thermolysin rapidly cleaved the linkage between the Fc and IL-33 region for all constructs, there were significant differences in the subsequent rate of degradation of the liberated IL-33 fragment (Fig. 7).

[0375] Based on production, stability and functional attributes, the candidate pool was further down-selected to focus on Bi_Fcl_ M14-IL-33 and Bi_Fcl_ M15-IL-33 variants. Monovalent equivalents of these two constructs, in which a single IL-33 moiety was fused to the 'knob' arm of KiH heterodimeric Fc fusions, were also produced for co-evaluation (Mo_Fcl_ M14-IL-33 and Mo_Fcl_M15-IL-33). These four constructs, together with the non-stabilized Bi_Fcl_Ml-IL-33, were assessed for stability after incubation in human serum at 37°C for up to 3 days (Fig. 8). Not unexpectedly, the non-stabilized Bi_Fcl_Ml-IL-33 was significantly degraded in serum. By contrast, no degradation was observed with monovalent and bivalent Ml 4-IL-33 -containing fusion proteins while minimal degradation was observed with monovalent and bivalent M15-IL-33-containing fusions (Fig. 8).

[0376] Based on these results, monovalent and bivalent M14-IL-33 fusions, containing a stabilizing disulfide bridge resulting from the H168C / V219C mutations, were moved forward.Atorney Docket No. MSKCC.064.WO1

[0377] Electronically filed on: January 9, 2026 EXAMPLE 8

[0378] Improvement Fc Glycosylation Pattern and Characterization of Aglycosylated Disulfide- Stabilized IL-33 Variants

[0379] Product heterogeneity due to variable glycosylation of an antibody or protein-based therapeutic can complicate the manufacturing process and potentially impact therapeutic efficacy. Having eliminated potential N-glycosylation motifs in the IL-33 portion of the fusion molecule, the lone remaining N-glycosylation site in Mo_Fcl_M14-IL-33 and Bi_Fcl_M14-IL-33 was atN297 within the Fc region. Intact mass analysis of these fusion proteins identified species that were consistent with occupancy of this glycosylation site by GOF (+ 1445 Da) and GIF (+ 1607 Da) biantennary glycans that are typical for CHO cell-expressed therapeutic antibodies. However, significant levels of an additional glycoform was observed, where the mass adduct (+ 1217 Da) was consistent with the high mannose glycan Man5 (Fig. 9). This was unrelated to the engineered disulfide in the IL-33 moiety as the non-stabilized Bi_Fcl_Ml-IL-33 was also found to have high levels of Man5 glycan.

[0380] The high levels of Man5 glycan observed on these Fc fusion proteins stands in contrast to the very low levels that are typically observed for therapeutic antibody products. High mannose glycan have been reported to have a deleterious impact on the pharmacokinetics of glycoproteins, often leading to a decrease in circulating half-life

[0018] , Given the increased N297 glycan heterogeneity and elevated Man5 level relative to antibody therapeutics, a mutation was designed (N297G) to completely eliminate N-linked glycosylation. Mo_aFcl_M14-IL-33 and Bi_aFcl_M14-IL-33 are the two resulting aglycosylated mono- and bivalent fusion proteins with N297G and L234A / L235A / P331 S mutations. Since removal of the N-linked glycosylation site is known to effectively silence Fc effector function

[0019] , mutations that had been previously introduced to abolish effector function (L234A / L235A / P331 S) were made redundant, therefore these residues were reverted back to wild-type. Based upon these 4 amino acid changes in the Fc region, the aglycosylated mono- and bivalent fusion proteins Mo_aFc_M14-IL-33 and Bi_aFc_M14-IL-33 were generated.

[0381] EXAMPLE 9

[0382] Production Attributes and Potency of Aglycosylated Disulfide-Stabilized IL-33 Variants

[0383] Parental Bi_aFcl_Ml-IL-33, Mo_aFcl_M14-IL-33, Bi_aFcl_M14-IL-33, Mo_aFc_M14-IL-33Atorney Docket No. MSKCC.064.WO1

[0384] Electronically filed on: January 9, 2026 and Bi_aFc_M14-IL-33 were recombinantly produced in CHO-express cells and purified. The functional activity of these fusions was assessed in the HEK-Blue™ IL-33 reporter assay and compared to their glycosylated precursors (Fig. 10). The comparable bioactivity for all of these samples indicated that removal of theN-linked glycan in the Fc had no impact on IL-33 bioactivity. At first glance, the production yield for aglycosylated constructs yield appeared superior to that of their glycosylated counterparts, but this difference was likely due to a switch in expression system rather than changes to the molecule (Fig. 10). In addition, the removal of the LALA / PS mutations also improved the level of production of Mo_aFc_M14-IL-33 and Bi_aFc_M14-IL-33 (Fig. 10).

[0385] Based on these results, Mo_aFc_M14-IL-33 and Bi_aFc_M14-IL-33 were moved forward.

[0386] EXAMPLE 10

[0387] Aglycosylated Di sulfide- Stabilized IL-33 Fc Fusion Proteins Exhibit Enhanced Serum Stability

[0388] To also ensure that aglycosylated variants were not compromised with respect to their stability in human serum, Mo_aFc_M14-IL-33 and Bi_aFc_M14-IL-33, together with the non-stabilized Bi_Fcl_Ml-IL-33, were assessed in the serum incubation assay (Fig.11). As previously observed, the non-stabilized Bi_Fcl_Ml-IL-33 was significantly degraded after incubation in serum. By contrast, a lower level of degradation was observed for aglycosylated Bi_aFc_M14-IL-33 when incubated for up to 72 h. Interestingly, degradation of the monovalent Mo_aFc_M14-IL-33 was much lower, though this may partly reflect the difference in concentration of IL-33 units, which is only half that of the corresponding bivalent format. Nevertheless, both mono- and bivalent aglycosylated candidates exhibited favorable characteristics.

[0389] EXAMPLE 11

[0390] Efficacy in Pancreatic Ductal Adenocarcinoma (PDAC) Mouse Model

[0391] To assess the impact of drug valency of Mo_aFcl_M14-IL-33 versus Bi_aFcl_M14-IL-33 on potency , an in vivo efficacy study was performed using a pancreatic ductal adenocarcinoma (PDAC) mouse model. Briefly, orthotopic tumor PDAC bearing mice were treated every other day for 4 weeks with either low or high dose of Mo_aFcl_M14-IL-33 and Bi_aFcl_M14-IL-33 (0.04 or 1.14 nmol / kg respectively). Both monovalent and bivalent variants showed an improved overall survival, reduction of tumor growth in a dose dependent manner, and an increase in the intratumoral KLRGl+ILC2s frequency and TLS densities, minimal potency difference was observed between monovalent and bivalent forms (Fig. 12). Since potential avidity effects due toAtorney Docket No. MSKCC.064.WO1

[0392] Electronically filed on: January 9, 2026 bivalency could potentially lead to excessive activity, thereby increasing safety risks, the monovalent format was prioritized.

[0393] To characterize the antitumor effect of the monovalent format: Mo_aFc_M14-IL-33, we performed a dose efficacy study. Orthotopic PDAC mice were randomized into 5 groups treated with a range of increasing doses of stabilized Mo_aFc_M14-IL-33 (doses were 0.0008, 0.004, 0.02, 0.1 and 0.5mg / kg). An Fc isotype control was used as a negative control (0.5mg / kg). The therapeutic and negative control treatment were administered intraperitoneally 3 times a week for 4 weeks. In addition, mouse recombinant IL-33 (mrL33) was used as a positive control, 0.025mg / kg was administered daily for the 1stweek then 3 times a week for the duration of the experiment. We found that Mo_aFc_M14-IL-33 exhibited tumor control even at the lower doses (0.004mg / kg) as compared to the untreated group. Surprisingly, this anti-tumor effect was even greater than the mouse recombinant IL-33 (mrIL-33). Consistently, monovalent stabilized Mo_aFc_M14-IL-33 expanded intratumoral ILC2s and formed TLSs in treated mice to a greater degree than the isotype control (Fig. 13).

[0394] EXAMPLE 12

[0395] Primate Study

[0396] Mo_aFcl_M14-IL-33 binds to the human ST2 receptor (Kd = 0.06 nM), and is cross-reactive with cynomolgus monkey ST2 (Kd = 0.22 nM), which shares 92.8% homology with human ST2. As such, we used cynomolgus monkeys (n=2; 1 male and 1 female) to assess the pharmacokinetic (PK) profile of Mo_aFcl_M14-IL-33 (two doses of 0.04 mg / kg hIL33 in PBS IV bolus on days 0 and day 7). Blood sampling was taken pre-dose, and various time points post-dose one on day 0 (10 min, 1 h, 8 h, 24 h, 48 h, 72 h, 120 h, and 144 h), pre dose two on day 7, and at various time points post dose two on day 7 (post second dose (10 min, 1 h, 8 h, 24 h, 48 h, 72 h, 120 h, 144 h, and 168 h).

[0397] The pharmacokinetic data is included in Tables 4 and 5 below. To quantify antibody concentration at each timepoint, we used anti -human IL-33 + human Fc ELISA with a sensitive electrochemiluminescence detection. The Cmax (the peak concentration in the bloodstream) reached 915 ng / mL and 997 ng / mL for the male and female cynomolgus monkeys, respectively. The baseline half-life of Mo_aFcl_M14-IL-33 following a single dose was 5.47 h and 39 h, respectively, for two individual animals. The lower limit of quantitation (LLOQ) of the electrochemiluminescence detection method was not sufficiently low for all terminal timepoints;Atorney Docket No. MSKCC.064.WO1

[0398] Electronically filed on: January 9, 2026 thus, the accuracy of the calculated half-life after the first dose may be limited. Following the second dose, the half-life stabilized at 11.4 h for both animals (Table 5), indicating stability of the molecule in vivo. Additionally, we checked for the presence of neutralizing antibody-drug antibodies (ADA) in serum using Mo_aFcl_M14-IL-33 + anti-monkey IgG ELISA with Tetramethylbenzidine (TMB) detection. While AD As were detected starting on day 13 following the first dose, systemic drug exposure (AUC 0-t) increased over time, which suggests that Mo_aFcl_M14-IL-33 is highly stable and potent.

[0399] Table 4: PK Summary Dose 1 (0.04 mg / kg)

[0400]

[0401] Table 5: PK Summary Doses 1 and 2 (0,04 mg / kg on days 0 and 7)

[0402]

[0403] Atorney Docket No. MSKCC.064.WO1

[0404] Electronically filed on: January 9, 2026 In Tables 4 & 5, the abbreviated terms having the following meanings: tU (h) is the elimination half-life (the time required for the plasma drug concentration to decrease by 50%); Cmax (ng / mL) is the maximum plasma concentration in ng / mL after dosing; AUCo-t (h*ng / mL) is the area under the concentration-time curve from 0 to t representing observed drug exposure from dosing (time 0) to the last measurable concentration (t); AUCo-co (h*ng / mL) is the area under the concentration-time curve from 0 to infinity, representing total systemic drug exposure; CL obs (mL / day / kg) is observed systemic clearance - the volume of plasma cleared of drug per unit time per kg body weight; MRTco,obs (h) is the mean residence time to infinity (observed); Vss_obs (mL / kg) is the observed volume of distribution at steady state and describes the extent of drug distribution in the body at steady state; Cmin (ng / mL) is the minimum (trough) plasma concentration; Cavg (ng / mL) is the average plasma concentration; Fluctuation%_Tau (%) is the percent fluctuation over the dosing interval; AUC TAU (h*ng / mL) is area under the concentration-time curve over one dosing interval and represents drug exposure during a single dosing interval (TAU) at steady state.

[0405] References

[0406] 1. Cayrol, C. and J.P. Girard, IL-33: an alarmin cytokine with crucial roles in innate immunity, inflammation and allergy. Curr Opin Immunol, 2014. 31: p. 31-7.

[0407] 2. Lefrancais, E., et al., IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G. Proc Natl Acad Sci U S A, 2012. 109(5): p. 1673-8.

[0408] 3. Luthi, A.U., et al., Suppression of interleukin-33 bioactivity through proteolysis by apoptotic caspases. Immunity, 2009. 31(1): p. 84-98.

[0409] 4. Ali, S., et al., IL-1 receptor accessory protein is essential for IL-33-induced activation of T lymphocytes and mast cells. Proc Natl Acad Sci U S A, 2007. 104(47): p. 18660-5. 5. Chackerian, A. A., et al., IL-1 receptor accessory protein and ST2 comprise the IL-33 receptor complex. J Immunol, 2007. 179(4): p. 2551-5.

[0410] 6. Cohen, E.S., et al., Oxidation of the alarmin IL-33 regulates ST2-dependent inflammation. Nat Commun, 2015. 6: p. 8327.

[0411] 7. Chen, W.Y., et al., Myocardial pressure overload induces systemic inflammation through endothelial cell IL-33. Proc Natl Acad Sci U S A, 2015. 112(23): p. 7249-54.

[0412] 8. Sanada, S., et al., IL-33 and ST2 comprise a critical biomechanically induced and cardioprotective signaling system. J Clin Invest, 2007. 117(6): p. 1538-49.

[0413] 9. Dominguez, D., et al., Exogenous IL-33 Restores Dendritic Cell Activation and Maturation in Established Cancer. J Immunol, 2017. 198(3): p. 1365-1375.Atorney Docket No. MSKCC.064.WO1

[0414] Electronically filed on: January 9, 2026 10. Eissmann, M.F., et al., Interleukin 33 Signaling Restrains Sporadic Colon Cancer in an Interferon-gamma-Dependent Manner. Cancer Immunol Res, 2018. 6(4): p. 409-421.

[0415] 11. Villarreal, D.O., et al., Alarmin IL-33 acts as an immunoadjuvant to enhance antigenspecific tumor immunity. Cancer Res, 2014. 74(6): p. 1789-800.

[0416] 12. Gao, X., et al., Tumoral expression of IL-33 inhibits tumor growth and modifies the tumor microenvironment through CD8+ T andNK cells. J Immunol, 2015. 194(1): p.

[0417] 438-45.

[0418] 13. Hollande, C., et al., Inhibition of the dipeptidyl peptidase DPP4 (CD26) reveals IL-33- dependent eosinophil-mediated control of tumor growth. Nat Immunol, 2019. 20(3): p.

[0419] 257-264.

[0420] 14. Lucarini, V., et al., IL-33 restricts tumor growth and inhibits pulmonary metastasis in melanoma-bearing mice through eosinophils. Oncoimmunology, 2017. 6(6): p. el317420.

[0421] 15. Moral, J. A., et al., ILC2s amplify PD-1 blockade by activating tissue-specific cancer immunity. Nature, 2020. 579(7797): p. 130-135.

[0422] 16. Amisaki, M., et al., IL33 activated ILC2s induce tertiary lymphoid structures in pancreatic cancer. Nature, In press.

[0423] 17. Kennard, M.L., et al., Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells. Biotechnol Bioeng, 1993. 42(4): p. 480-6.

[0424] 18. Boune, S., et al., Principles ofN-Linked Glycosylation Variations oflgG-Based Therapeutics: Pharmacokinetic and Functional Considerations. Antibodies (Basel), 2020. 9(2).

[0425] 19. Ju, M.S. and S.T. Jung, Aglycosylated full-length IgG antibodies: steps toward nextgeneration immunotherapeutics. Curr Opin Biotechnol, 2014. 30: p. 128-39.

[0426] 0. Salam, N.K., et al., Structure-based approach to the prediction of disulfide bonds in proteins. Protein Eng Des Sei, 2014. 27(10): p. 365-74.

[0427] 1. Alt, C., et al., Long-Acting IL-33 Mobilizes High-Quality Hematopoietic Stem and Progenitor Cells More Efficiently Than Granulocyte Colony-Stimulating Factor or AMD3100. Biol Blood Marrow Transplant, 2019. 25(8): p. 1475-1485.

Claims

Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 CLAIMS1. A recombinant IL-33 protein that comprises at least one IL-33 domain, wherein the IL- 33 domain comprises a variant of mature wild-type human IL-33 that comprises: a S173A mutation, a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation.

2. A recombinant IL-33 protein according to claim 1, wherein the IL 33 domain further comprises the pair of “to cysteine” mutations H168C and V219C.

3. A recombinant IL-33 protein according to claim 2, wherein the IL-33 domain comprises SEQIDNO. 19.

4. A recombinant IL-33 protein that is an Fc fusion protein comprising: (a) an IL-33 domain comprising SEQ ID NO. 19 and (b) an Fc domain.

5. A recombinant IL-33 protein according to claim 4, wherein the Fc fusion protein is a bivalent dimer comprising a first chain and a second chain, wherein the first chain and the second chain each comprise SEQ ID NO. 52.

6. A recombinant IL-33 protein according to claim 4, wherein the Fc fusion protein is a monovalent dimer comprising a first chain and a second chain, wherein the first chain comprises SEQ ID NO. 53 and the second chain comprises SEQ ID NO. 54.

7. A nucleic acid molecule that encodes a recombinant IL-33 protein according to any one of claims 1-4 or that encodes a first chain or a second chain of a dimer according to claim 5 or claim 6.

8. A vector that comprises a nucleic acid molecule that encodes a recombinant IL-33 protein according to any one of claims 1-4 or that encodes a first chain or a second chain of a dimer according to claim 5 or claim 6.

9. A host cell that comprises a nucleic acid molecule according to claim 7 or a vector according to claim 8.

10. A pharmaceutical composition comprising a recombinant IL-33 protein according to any one of claims 1-6 and at least one pharmaceutically acceptable carrier orexcipient.Atorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 11. A method of activating an IL-33 receptor, the method comprising contacting an IL- 33 receptor, in vitro or in vivo, with a recombinant IL-33 protein according to any one of claims 1-6.

12. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject, the method comprising administering a recombinant IL-33 protein according to any one of claims 1-6 to a subject in need thereof.

13. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject, the method comprising administering a pharmaceutical composition according to claim 10 to a subject in need thereof.

14. A recombinant IL-33 protein comprising an IL-33 domain, wherein the IL-33 domain is a variant of mature wild-type human IL-33 that comprises:(i) at least one pair of “to cysteine” mutations selected from:(a) P118C and S162C,(b) L123C and K158C,(c) S127C and K266C,(d) Y129C and L264C,(e) I134C and V147C,(f) F136C and D157C,(g) V147C and V242C,(h) L161C and V184C,(i) S162C and T185C,(j) Y164C and M183C,(k) S166C and Ml 83 C,(l) S166C and V219C,(m)H168C and V219C,(n) S170C and G179C,(o) N171C and G179C,(p) A196C and Q215C,(q) V203C and I240C,(r) V203C and L247C,(s) Q215C and T234C,Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 (t) S229C and 1263 C,(u) F239C and 1263 C,(v) G241C and N262C; and(w)G241C and 1263 C;or(ii) the pair of cysteines A196C and C232, wherein the cysteine at amino acid residue 196 is a “to cysteine” mutation.

15. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises:(i) at least one pair of “to cysteine” mutations selected from:(a) L123C and K158C;(b) Y129C and L264C;(c) F136C and D157C;(d) V147C and V242C;(e) S166C and M183C;(f) H168C and V219C;(g) S170C and G179C;(h) N171C and G179C;(i) Q215C and T234C;(j) G241C and N262C; and(k) G241C and 1263 C;or(ii) the pair of cysteines A196C and C232, wherein the cysteine at amino acid residue 196 is a “to cysteine” mutation.

16. A recombinant IL-33 protein according to claim 15, wherein the IL-33 domain comprises at least one pair of “to cysteine” mutations selected from:(a) L123C and K158C;(b) H168C and V219C;(c) S170C and G179C; and(d) N171C and G179C.Atorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 17. A recombinant IL-33 protein according to claim 16, wherein the IL-33 domain comprises at least one pair of “to cysteine” mutations selected from:(a) H168C and V219C; and(b) S170C and G179C.

18. A recombinant IL-33 protein according to claim 17, wherein the IL-33 domain comprises the pair of “to cysteine” mutations: H168C and V219C.

19. A recombinant IL-33 protein according to any one of claims 14-18, wherein the IL-33 domain further comprises:(a) a S173 A mutation, and(b) either (i) a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, or (ii) a C208S mutation, a C227A mutation, and a C259A mutation.

20. A recombinant IL-33 protein according to any one of claims 14-18, wherein:(i) the IL-33 domain further comprises a S173A mutation, a C208S mutation, a C227A mutation, a C232S mutation, and a C259A mutation, or(ii) if the IL-33 domain comprises a “to cysteine” mutation A196C and a cysteine at amino acid residue 232 (C232), the IL-33 domain further comprises a SI 73 A mutation, a C208S mutation, a C227A mutation, and a C259A mutation.

21. A recombinant IL-33 protein according to any one of claims 14-20, wherein the IL-33 domain further comprises at least one intramolecular disulfide bond.

22. A recombinant IL-33 protein according to claim 21, wherein the at least one intramolecular disulfide bond connects the two cysteines in the pair of “to cysteine” mutations or connects the pair of cysteines A196C and C232.

23. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 7-30.

24. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 8, 10, 12, 13, 17, 19, 20, 21, 23, 26, 29 and 30.Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 25. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 8, 19, 20, and 21.

26. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises the amino acid sequence any one of SEQ ID NOs 19 and 20.

27. A recombinant IL-33 protein according to claim 14, wherein the IL-33 domain comprises the amino acid sequence of SEQ ID NO. 19.

28. A recombinant IL-33 protein according to any one of claims 14-27, wherein the recombinant IL-33 protein is an Fc fusion protein and further comprises a human Fc domain.

29. A recombinant IL-33 protein according to claim 28, wherein the Fc domain is an IgGl, IgG2, IgG3, or IgG4 Fc domain.

30. A recombinant IL-33 protein according to claim 29, wherein the Fc domain is an IgGl or IgG4 Fc domain.

31. A recombinant IL-33 protein according to claim 28, wherein the Fc domain is an IgGl Fc domain.

32. A recombinant IL-33 protein according to any one of claims 28-31, wherein the Fc domain is a variant of a wild-type human IgG Fc domain that comprises one or more mutations that reduce or eliminate Fc effector function.

33. A recombinant IL-33 protein according to any one of claims 28-32, wherein the Fc domain comprises is a variant of a wild-type human IgGl Fc domain that comprises one or more of the following mutations: L234A, L235A, P331 S, and / or N297G.

34. A recombinant IL-33 protein according to claim 33, wherein the Fc domain comprises each of: a L234A mutation, a L235A mutation, a P33 IS mutation, and an N297G mutation.

35. A recombinant IL-33 protein according to claim 33, wherein the Fc domain comprises each of: a L234A mutation, a L235 A mutation, and a P331 S mutation.Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 36. A recombinant IL-33 protein according to claim 33, wherein the Fc domain comprises an N297G mutation.

37. A recombinant IL-33 protein according to claim 33, wherein the Fc domain comprises an N297G mutation and does not comprise an L234A, L235A, or P331 S mutation.

38. A recombinant IL-33 protein according to any one of claims 28-30 or 32, wherein the Fc domain comprises is a variant of a wild-type human IgG4 Fc domain that comprises one or more of the following mutations: F234A, L235A, S228P and / or N297G.

39. A recombinant IL-33 protein according to claim 38, wherein the Fc domain comprises a F234A mutation, a L235A mutation, and a S228P mutation.

40. A recombinant IL-33 protein that is an Fc fusion protein comprising an IL-33 domain and an Fc domain, wherein the IL-33 domain comprises any one of SEQ ID NOs 4-30 and wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NOs. 33- 65.

41. A recombinant IL-33 protein according to claim 40, wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NOs. 33-49, 52, 55, 58 and 61-65.

42. A recombinant IL-33 protein according to claim 40, wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NOs. 50-51, 53-54, 56-57, 59-60.

43. A recombinant IL-33 protein according to claim 40, wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NOs. 50, 53, 56, or 59.

44. A recombinant IL-33 protein according to claim 40, wherein the Fc domain comprises the Fc domain portion of any one of SEQ ID NOs. 51, 54, 57, or 60.

45. A recombinant IL-33 protein according to any one of claims 28-44, wherein the Fc domain is located N-terminal to the IL-33 domain.

46. A recombinant IL-33 protein according to any one of claims 28-44, wherein the Fc domain is located C-terminal to the IL-33 domain.

47. A recombinant IL-33 protein according to any one of claims 28-46, wherein the IL-33 domain is connected to the Fc domain by a peptide linker.Atorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 48. A recombinant IL-33 protein according to claim 47, wherein the peptide linker is a G / S linker.

49. A recombinant IL-33 protein according to claim 47, wherein the peptide linker is a (GxS)nlinker, where x is from 1-10 and n is from 1 to 10.

50. A recombinant IL-33 protein according to claim 47, wherein the peptide linker is a (628)3 linker, a (638)3 linker, or a (648)3 linker.

51. A recombinant IL-33 protein according to claim 28, comprising any one of SEQ ID NOs.NO. 33-65.

52. A recombinant IL-33 protein according to claim 28, comprising any one of SEQ ID NOs.33-49, 52, 55, 58 and 61-65.

53. A recombinant IL-33 protein according to claim 28, comprising any one of SEQ ID NOs.50-51, 53-54, 56-57, 59-60.

54. A recombinant IL-33 protein according to claim 28, comprising any one of SEQ ID NOs.50, 53, 56, or 59.

55. A recombinant IL-33 protein according to claim 28, comprising any one of SEQ ID NOs.51, 54, 57, or 60.

56. A recombinant IL-33 protein according to any one of claims 28-55, wherein the recombinant IL-33 protein is dimeric, comprising a first chain and a second chain.

57. A dimeric recombinant IL-33 protein according to claim 56, wherein the dimeric recombinant IL-33 protein is bivalent and the first chain and the second chain each comprise an IL-33 domain.

58. A dimeric recombinant IL-33 protein according to claim 56, wherein the dimeric recombinant IL-33 protein is monovalent wherein the first chain comprises an IL-33 domain and the second chain does not comprise an IL-33 domain.

59. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical and each comprise an Fc fusion protein according to any one of claims 28-55.Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 60. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical and each comprise any one of SEQ ID NOs. 33- 49, 52, 55, 58 and 61-65.

61. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprise any one of SEQ ID NOs. 38- 49, 52, 55, 58 and 61-65.

62. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises any one of SEQ ID NOs. 40-49, 52, 55, 58 and 61-65.

63. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises any one of SEQ ID NOs. 42-49, 52, 55, 58 and 61-65.

64. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises any one of NO. 44-49, 52, 55, 58 and 61-65.

65. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises any one of SEQ ID NOs. 48, 49, 52, 55, and 58.

66. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises any one of SEQ ID NOs. 48, 49, 52, and 55.

67. A bivalent dimeric recombinant IL-33 protein according to claim 57, wherein the first chain and the second chain are identical, and each comprises SEQ ID NO. 52.

68. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises an Fc fusion protein according to any of claims 28-55, and the second chain comprises an Fc domain but does not comprise an IL-33 domain.

69. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises any one of SEQ ID NOs. 50, 53, 56 and 59, and the second chain comprises any one of SEQ ID NOs 51, 54, 57 and 60.Atorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 70. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises SEQ ID NO. 50 and the second chain comprises SEQ ID NO. 51.

71. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises SEQ ID NO. 53 and the second chain comprises SEQ ID NO. 54.

72. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises SEQ ID NO. 56 and the second chain comprises SEQ ID NO. 57.

73. A monovalent dimeric recombinant IL-33 protein according to claim 58, wherein the first chain comprises SEQ ID NO. 59 and the second chain comprises SEQ ID NO. 60.

74. A nucleic acid molecule that encodes a recombinant IL-33 protein according to any one of claims 14-73.

75. A vector that comprises a nucleic acid molecule that encodes a recombinant IL-33 protein according to any one of claims 14-73.

76. A host cell that comprises a nucleic acid molecule according to claim 74 or a vector according to claim 75.

77. A pharmaceutical composition comprising a recombinant IL-33 protein according to any one of claims 14-73 and at least one pharmaceutically acceptable carrier or excipient.

78. A method of activating an IL-33 receptor, the method comprising contacting an IL-33 receptor, in vitro or in vivo, with a recombinant IL-33 protein according to any one of claims 14-73 or a pharmaceutical composition according to claim 77.

79. A method of expanding or activating ILC2 cells, the method comprising contacting ILC2 cells, in vitro or in vivo, with a recombinant IL-33 protein according to any one of claims 14-73 or a pharmaceutical composition according to claim 77.

80. A method of treating a condition amenable to treatment with IL-33 or an IL-33 receptor agonist in a subject, the method comprising administering a recombinant IL-33 protein according to any one of claims 14-73 or a pharmaceutical composition according to claim 77 to a subject in need thereof.Attorney Docket No. MSKCC.064.WO1Electronically filed on: January 9, 2026 81. A method of treating cancer in a subject, the method comprising administering a recombinant IL-33 protein according to any one of claims 14-73 or a pharmaceutical composition according to claim 77 to a subject in need thereof.

82. A method of treating pancreatic ductal adenocarcinoma (PDAC) in a subject, the method comprising administering a recombinant IL-33 protein according to any one of claims 14- 73 or a pharmaceutical composition according to claim 77 to a subject in need thereof.

83. A method of treating immune checkpoint inhibitor resistant pancreatic ductal adenocarcinoma (PDAC) in a subject, the method comprising administering a recombinant IL-33 protein according to any one of claims 14-73 or a pharmaceutical composition according to claim 77 to a subject in need thereof.

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