WO2023164286A1

WO2023164286A1 - Engineered cd122 compositions and methods thereof

Info

Publication number: WO2023164286A1
Application number: PCT/US2023/014117
Authority: WO
Inventors: Dheeraj TOMAR; Ertan Eryilmaz
Original assignee: Xilio Development, Inc.
Priority date: 2022-02-28
Filing date: 2023-02-28
Publication date: 2023-08-31

Abstract

The present invention relates to an engineered CD 122 or a fragment thereof, which has improved stability as compared to a wild-type CD122. The engineered CD122 of the present invention can be incorporated into a masked cytokine comprising a cytokine, an engineered CD 122, Fc domain, and a cleavable linker. The engineered CD 122 of the present invention can also be incorporated into a targeting cytokine comprising a cytokine, a targeting moiety, an engineered CD122, Fc domain, and a cleavable linker. The targeting moiety comprises an antigen-binding moiety that specifically binds to an antigen expressed on the surface of a target cell. CD 122 masks the IL-2 or IL- 15 cytokine or functional fragment thereof thereby reducing or preventing binding of the cytokine or functional fragment thereof to its cognate receptor, but upon proteolytic cleavage of the cleavable linker at a target site, the cytokine or functional fragment thereof becomes activated, which renders it capable or more capable of binding to its cognate receptor.

Description

ENGINEERED CD122 COMPOSITIONS AND METHODS THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, U.S. provisional application No. 63/314,944, filed on February 28, 2022, the contents of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Cancer is the second leading cause of death in the United States, accounting for more deaths than the next five leading causes (chronic respiratory disease, stroke, accidents, Alzheimer’s disease and diabetes). While great strides have been made especially with targeted therapies, there remains a great deal of work that are yet to be done. Immunotherapy and a branch of this field, immuno-oncology, is creating viable and exciting therapeutic options for treating malignancies. Specifically, it is now recognized that one hallmark of cancer is immune evasion and significant efforts have identified targets and developed therapies to these targets to reactivate the immune system to recognize and treat cancer.

[0003] Cytokine therapy is an effective strategy for stimulating the immune system to induce anti-tumor cytotoxicity. In particular, aldesleukin, a recombinant form of interleukin-2 (IL-2), has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, cytokines that are administered to patients generally have a very short half-life, thereby requiring frequent dosing. For instance, the product label of aldesleukin, marketed under the brand name Proleukin, states that the drug was shown to have a half-life of 85 minutes in patients who received a 5-minute intravenous (IV) infusion. In addition, administration of high doses of cytokine can cause adverse health outcomes, such as vascular leakage, through systemic immune activation. These findings illustrate the need for developing cytokine therapeutics that effectively target tumors without the side effects associated with systemic immune activation.

Provided herein are engineered CD 122 masking moi eties, and compositions thereof and methods of use thereof for addressing the needs described above. SUMMARY OF THE INVENTION

[0004] The present invention provides, among other things, an engineered CD 122 (i.e., an engineered masking moiety), and compositions and methods for its use in treatment of cancer. The engineered CD122 comprises one or more mutations as compared to the wild-type CD122. The engineered CD122 binds to IL-2, IL-15, or any other cytokine that possess binding affinity to the engineered CD122. The present invention is based, in part, on the surprising discovery that masked IL-2 cytokines comprising the engineered CD 122 as a masking moiety possess an improved stability including improved thermal stability and stress tolerance ability.

[0005] The present invention further provides the PD-1 targeting masked IL-2 cytokines comprising the engineered CD122 as a masking moiety to specifically target a cell of interest for effective treatment of cancer without causing undesired side effects.

[0006] In one aspect, the present invention provides, among other things, an engineered CD122 polypeptide or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence.

[0007] In another aspect, the present invention provides, among other things, an engineered CD122 polypeptide or fragment thereof comprising a mutation at amino acid 122 and 168 relative to SEQ ID NO: 15, wherein the engineered CD122 has improved stability compared to a wild-type CD122.

[0008] In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 80% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 82% identity to SEQ ID NO: 15. In some embodiments, the engineered CD 122 polypeptide comprises an amino acid sequence with at least 85% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 87% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 91% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 92% identity to SEQ ID NO: 15. In some embodiments, the engineered CD 122 polypeptide comprises an amino acid sequence with at least 93% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 94% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 96% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 97% identity to SEQ ID NO: 15. In some embodiments, the engineered CD 122 polypeptide comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 15. In some embodiments, the engineered CD122 polypeptide comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 15.

[0009] In some embodiments, the engineered CD122 polypeptide comprises a mutation to facilitate disulfide bond formation. In some embodiments, the engineered CD122 polypeptide comprises a mutation that creates a salt bridge.

[0010] In some embodiments, the engineered CD122 polypeptide comprises F8C mutation. In some embodiments, the engineered CD122 polypeptide comprises A94C mutation. In some embodiments, the engineered CD122 polypeptide comprises L106C mutation. In some embodiments, the engineered CD122 polypeptide comprises C122S mutation. In some embodiments, the engineered CD122 polypeptide comprises C122V mutation. In some embodiments, the engineered CD122 polypeptide comprises C122A mutation. In some embodiments, the engineered CD122 polypeptide comprises N 123 C mutation. In some embodiments, the engineered CD122 polypeptide comprises N 123 Q mutation. In some embodiments, the engineered CD122 polypeptide comprises C168V mutation. In some embodiments, the engineered CD122 polypeptide comprises C168A mutation. In some embodiments, the engineered CD122 polypeptide comprises C168S mutation. In some embodiments, the engineered CD122 polypeptide comprises L169C mutation. In some embodiments, the engineered CD122 polypeptide comprises Q177C mutation. In some embodiments, the engineered CD122 polypeptide comprises V184C mutation. In some embodiments, the engineered CD122 polypeptide comprises S195C mutation. In some embodiments, the engineered CD 122 polypeptide comprises R204C mutation.

[0011] In some embodiments, the engineered CD122 polypeptide comprises C122S and C168S mutations. In some embodiments, the engineered CD122 polypeptide comprises Cl 22 A and N 123 C mutations.

[0012] In some embodiments, the engineered CD122 polypeptide comprises C122V and C168Vmutations. In some embodiments, the engineered CD122 polypeptide comprises C122A and C168V mutations. In some embodiments, the engineered CD122 polypeptide comprises C122V and C168A mutations. In some embodiments, the engineered CD122 polypeptide comprises C122V and N123C mutations. In some embodiments, the engineered CD122 polypeptide comprises C122A and C168A mutations. In some embodiments, the engineered CD122 polypeptide comprises VI 17C, N123Q and C168A mutations. In some embodiments, the engineered CD122 polypeptide comprises N 123 Q, C168A, and L169C mutations. In some embodiments, the engineered CD122 polypeptide 'comprises L106C, C122A, C168A and S195C mutations. In some embodiments, the engineered CD122 polypeptide comprises L106C, C122A, C168A and V184C mutations. In some embodiments, the engineered CD122 polypeptide comprises C122A, C168A, V184C, and S195C mutations. In some embodiments, the engineered CD122 polypeptide comprises C122A, C168A, Q177C, and R204C mutations. In some embodiments, the engineered CD122 polypeptide comprises L106C, C122V, C168V, and S195C mutations. In some embodiments, the engineered CD122 polypeptide comprises F8C, A94C, C122V, and C168V mutations.

[0013] In some embodiments, the improved stability is measured by percent high molecular weight species (HMWS). In some embodiments, the improved stability is measured by melting temperature.

[0014] In some embodiments, the engineered CD 122 polypeptide has less than 80

% of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 70 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 60 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 50 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 40 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 30 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 25 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 20% of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 15 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 12% of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 10 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 8 % of HMWS upon storage for two weeks at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 5 % of HMWS upon storage for two weeks at 37 °C.

[0015] In some embodiments, the engineered CD122 polypeptide has less than 70 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 60 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 50 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 40 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 30 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 20 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 10 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 8 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 7 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 6 % of HMWS upon storage for one week at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 5 % of HMWS upon storage for one week at 37 °C.

[0016] In some embodiments, the engineered CD 122 polypeptide has less than 10% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 9 % of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 8% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 7% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 6% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 5% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 4% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD122 polypeptide has less than 3% of HMWS upon storage for at least 3 days at 37 °C. In some embodiments, the engineered CD 122 polypeptide has less than 2% of HMWS upon storage for at least 3 days at 37 °C.

[0017] In some embodiments, the engineered CD 122 has a melting temperature of greater than 54 °C. In some embodiments, the engineered CD122 has a melting temperature of greater than 55 °C. In some embodiments, the engineered CD122 has a melting temperature of greater than 55.5 °C. In some embodiments, the engineered CD122 has a melting temperature of greater than 56 °C. In some embodiments, the engineered CD 122 has a melting temperature of greater than 56.5 °C. In some embodiments, the engineered CD122 has a melting temperature of greater than 57 °C. In some embodiments, the engineered CD122 has a melting temperature between 54.5-60°C. In some embodiments, the engineered CD122 has a melting temperature between 55-58°C. In some embodiments, the engineered CD122 has a melting temperature between 55.5- 57.5°C. In some embodiments, the engineered CD122 has a melting temperature between 56-57°C.

[0018] In some embodiments, the engineered CD122 polypeptide is fused to an Fc domain. In some embodiments, the engineered CD122 polypeptide is fused to a cytokine. In some embodiments, the engineered CD122 polypeptide is fused to a targeting moiety.

[0019] In one aspect, the present invention provides, among other things, a masked cytokine comprising a cytokine or a fragment thereof, a masking moiety comprising CD122 or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence, and a carrier moiety.

[0020] In some embodiments, the carrier moiety comprises a PEG molecule. In some embodiments, the carrier moiety comprises an albumin (e.g., a human serum albumin) or a fragment thereof. In some embodiments, the carrier moiety comprises an antibody. In some embodiments, the carrier moiety comprises an Fc domain. In some embodiments, the carrier moiety comprises an antibody or an antigen-binding fragment thereof.

[0021] In some embodiments, a masking moiety is linked to a cytokine or a fragment thereof, which is linked to a carrier moiety. In some embodiments, a masking moiety is linked to a carrier moiety, which is linked to a cytokine or a fragment thereof. In some embodiments, a masking moiety is linked to a cytokine or a fragment thereof via a cleavable linker, wherein the cytokine or a fragment thereof is linked to a carrier moiety via a non-cleavable linker. In some embodiments, a masking moiety is linked to a cytokine or a fragment thereof via a non-cleavable linker, wherein the cytokine or a fragment thereof is linked to a carrier moiety via a cleavable linker. In some embodiments, a masking moiety is linked to a cytokine or a fragment thereof via a cleavable linker, wherein the cytokine or a fragment thereof is linked to a carrier moiety via a cleavable linker. In some embodiments, a masking moiety is linked to a cytokine or a fragment thereof via a non-cleavable linker, wherein the cytokine or a fragment thereof is linked to a carrier moiety via a non-cleavable linker.

[0022] In some embodiments, a masking moiety is linked to a carrier moiety via a cleavable linker, wherein the carrier moiety is linked to a cytokine or a fragment thereof via a cleavable linker. In some embodiments, a masking moiety is linked to a carrier moiety via a non-cleavable linker, wherein the carrier moiety is linked to a cytokine or a fragment thereof via a cleavable linker. In some embodiments, a masking moiety is linked to a carrier moiety via a cleavable linker, wherein the carrier moiety is linked to a cytokine or a fragment thereof via a non-cleavable linker. In some embodiments, a masking moiety is linked to a carrier moiety via a non-cleavable linker, wherein the carrier moiety is linked to a cytokine or a fragment thereof via a non-cleavable linker. [0023] In one aspect, the present invention provides, among other things, a masked cytokine comprising a cytokine or a fragment thereof, a masking moiety comprising CD122 or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence, and an Fc domain.

[0024] In some embodiments, the Fc domain comprises a first Fc polypeptide linked to the masking moiety through a cleavable linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a cleavable linker. In some embodiments, the Fc domain comprises a first Fc polypeptide linked to the masking moiety through a non-cleavable linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a non-cleavable linker. In some embodiments, the Fc domain comprises a first Fc polypeptide linked to the masking moiety through a non- cleavable linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a cleavable linker. In some embodiments, the Fc domain comprises a first Fc polypeptide linked to the masking moiety through a cleavable linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a non-cleavable linker.

[0025] In one aspect, the present invention provides, among other things, a masked cytokine comprising a cytokine or a fragment thereof, a masking moiety comprising CD122 or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence, and an Fc domain comprising a first Fc polypeptide linked to the masking moiety through a first linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a second linker, wherein the masking moiety binds to the cytokine or fragment thereof; and wherein the first or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage.

[0026] In some embodiments a non-cleavable linker comprises

GGSSPPGGGSSGGGSG. [0027] In some embodiments, the cleavable linker comprises MPYDLYHP. In some embodiments, the cleavable linker comprises VPLSLYSG. In some embodiments, the cleavable linker comprises MPYDLYHP and VPLSLYSG. In some embodiments, the cleavable linker comprises DSGGFMLT. In some embodiments, the cleavable linker comprises HEQLTV. In some embodiments, the cleavable linker comprises RAAAVKSP. In some embodiments, the cleavable linker comprises DLLAVVAAS. In some embodiments, the cleavable linker comprises ISSGLLSGRS.

[0028] In some embodiments, the cleavable linker comprises between 6-60 amino acid residues. In some embodiments, the cleavable linker comprises between 8-50 amino acid residues. In some embodiments, the cleavable linker comprises between 8-30 amino acid residues. In some embodiments, the cleavable linker comprises between 8-25 amino acid residues. In some embodiments, the cleavable linker comprises 8 amino acid residues. In some embodiments, the cleavable linker comprises 9 amino acid residues. In some embodiments, the cleavable linker comprises 10 amino acid residues. In some embodiments, the cleavable linker comprises 11 amino acid residues. In some embodiments, the cleavable linker comprises 12 amino acid residues. In some embodiments, the cleavable linker comprises 13 amino acid residues. In some embodiments, the cleavable linker comprises 14 amino acid residues. In some embodiments, the cleavable linker comprises 15 amino acid residues. In some embodiments, the cleavable linker comprises 16 amino acid residues. In some embodiments, the cleavable linker comprises 17 amino acid residues. In some embodiments, the cleavable linker comprises 18 amino acid residues. In some embodiments, the cleavable linker comprises 19 amino acid residues. In some embodiments, the cleavable linker comprises 20 amino acid residues. In some embodiments, the cleavable linker comprises 21 amino acid residues. In some embodiments, the cleavable linker comprises 22 amino acid residues. In some embodiments, the cleavable linker comprises 23 amino acid residues. In some embodiments, the cleavable linker comprises 24 amino acid residues. In some embodiments, the cleavable linker comprises 25 amino acid residues.

[0029] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on the N-terminus by a spacer domain. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on the C- terminus by a spacer domain. In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide flanked on both sides by a spacer domain. In some embodiments, the spacer domain is between 2 and 20 amino acids in length. In some embodiments, the spacer domain is between 3 and 10 amino acids in length. In some embodiments, the spacer domain is between 3 and 6 amino acids in length. In some embodiments, the spacer domain is 2 amino acids in length. In some embodiments, the spacer domain is 3 amino acids in length. In some embodiments, the spacer domain is 4 amino acids in length. In some embodiments, the spacer domain is 5 amino acids in length. In some embodiments, the spacer domain is 6 amino acids in length. In some embodiments, the spacer domain is 7 amino acids in length. In some embodiments, the spacer domain is 8 amino acids in length. In some embodiments, the spacer domain is 9 amino acids in length. In some embodiments, the spacer domain is 10 amino acids in length. In some embodiments, the spacer domain is 11 amino acids in length. In some embodiments, the spacer domain is 12 amino acids in length. In some embodiments, the spacer domain is 13 amino acids in length. In some embodiments, the spacer domain is 14 amino acids in length. In some embodiments, the spacer domain is 15 amino acids in length.

[0030] In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding about 1-99% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding about 5-80% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding about 8- 60% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding about 10-40% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding about 10-20% active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 3 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 5 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 8 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 10 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 12 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 15 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 20 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 25 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 30 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 35 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 40 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 45 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 50 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 60 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 70 % active cytokine. In some embodiments, the cleavable linker has an in vitro cleavage efficiency yielding at least 80 % active cytokine.

[0031] In some embodiments, the first linker is the cleavable linker. In some embodiments, the second linker is the cleavable linker. In some embodiments, both the first linker and the second linker comprise a cleavable linker.

[0032] In some embodiments, a cytokine or a fragment there of is IL-2. In some embodiments, the IL-2 is a modified IL-2 cytokine or functional fragment thereof compared to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications R38A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications F42A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications Y45A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications E62A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications R38A, F42A, Y45A, and E62A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises the modification C125A relative to the sequence of a mature IL-2 having SEQ ID NO: 13.

[0033] In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises modifications R38A, F42A, Y45A, and E62A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. In some embodiments, the modified IL-2 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 14.

[0034] In some embodiments, a cytokine or a fragment there of is IL-15. In some embodiments, the IL-15 polypeptide comprises an amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions E46 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions E53 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions N71 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions N79 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions N112 as compared to the amino acid sequence of SEQ ID NO: 93.

[0035] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, or E53 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N79 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, or N112 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 94.

[0036] In some embodiments, the masked cytokine further comprises a targeting moiety.

[0037] In one aspect, the present invention provides, among other things, a targeted cytokine comprising a targeting moiety, a cytokine or a fragment thereof, an engineered CD122 masking moiety, and an Fc domain comprising a first Fc polypeptide linked to the cytokine or a fragment thereof through a first linker and a second Fc polypeptide linked to the masking moiety through a second linker.

[0038] In some embodiments, the CD122 is an engineered CD122 polypeptide or a fragment thereof comprising one or more mutations relative to a wild-type CD122 amino acid sequence.

[0039] In some embodiments, the engineered CD 122 polypeptide or a fragment thereof comprises a mutation at amino acid 122 and 168 relative to SEQ ID NO: 15, wherein the engineered CD122 has improved stability compared to a wild-type CD122.

[0040] In some embodiments, the targeting moiety comprises at least one antigen binding domain. In some embodiments, the targeting moiety comprises a first antigen binding domain and a second antigen binding domain. In some embodiments, the targeting moiety comprises a first antigen binding domain, a second antigen binding domain, and a third antigen binding domain. In some embodiments, the targeting moiety comprises a first antigen binding domain, a second antigen binding domain, a third antigen binding domain, and a fourth antigen binding domain. In some embodiments the first and second antigen binding domains specifically bind to the same target. In some embodiments the first, second, and a third antigen binding domains specifically bind to the same target. . In some embodiments the first, second, third, and fourth antigen binding domains specifically bind to the same target. In some embodiments the first and second antigen binding domains specifically bind to different targets. In some embodiments the first, second, and third antigen binding domains each specifically bind to different targets. In some embodiments the first, second, third, and fourth antigen binding domains each specifically bind to different targets.

[0041] In some embodiments, the first and second antigen binding domains comprise different amino acid sequences. In some embodiments, the first and second antigen binding domains comprise same amino acid sequences. In some embodiments, the first, second, and third antigen binding domains comprise different amino acid sequences. In some embodiments, the first, second, and third antigen binding domains comprise same amino acid sequences. In some embodiments, the first, second, third, and fourth, antigen binding domains comprise different amino acid sequences. In some embodiments, the first, second, third, and fourth antigen binding domains comprise same amino acid sequences.

[0042] In some embodiments, the first Fc polypeptide comprises a first IgGl Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG2 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG3 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises a first IgG4 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgGl Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG2 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG3 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises a second IgG4 Fc domain or a fragment thereof.

[0043] In some embodiments, the first Fc domain contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptide. In some embodiments, the second Fc domain contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptide. In some embodiments, the first and or the second Fc domains each contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptide. In some embodiments, the first and the second Fc domains each contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptide.

[0044] In some embodiments, the first Fc domain comprises Y349C mutation. In some embodiments, the first Fc domain comprises T366S mutation. In some embodiments, the first Fc domain comprises L368A mutation. In some embodiments, the first Fc domain comprises Y407V mutation. In some embodiments, the first Fc domain comprises N297A mutation. In some embodiments, the first Fc domain comprises S354C mutation. In some embodiments, the first Fc domain comprises T366W mutation. In some embodiments, the first Fc domain comprises 1253 A mutation. In some embodiments, the second Fc domain comprises Y349C mutation. In some embodiments, the second Fc domain comprises T366S mutation. In some embodiments, the second Fc domain comprises L368A mutation. In some embodiments, the second Fc domain comprises Y407V mutation. In some embodiments, the second Fc domain comprises N297A mutation. In some embodiments, the second Fc domain comprises S354C mutation. In some embodiments, the second Fc domain comprises T366W mutation. In some embodiments, the second Fc domain comprises I253A mutation.

[0045] In some embodiments, the first Fc domain comprises Y349C and T366S mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, and L368A mutations. In some embodiments, the first Fc domain comprises Y349C and L368A mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, and Y407V mutations. In some embodiments, the first Fc domain comprises T366S, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, L368A, and Y407V mutations. In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations. In some embodiments, the first Fc domain comprises S354C and T366W mutations. In some embodiments, the first Fc domain comprises S354C and N297A mutations. In some embodiments, the first Fc domain comprises T366W and N297A mutations. In some embodiments, the first Fc domain comprises S354C, T366W and N297A mutations. In some embodiments, the second Fc domain comprises Y349C and T366S mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, and L368A mutations. In some embodiments, the second Fc domain comprises Y349C and L368A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, and Y407V mutations. In some embodiments, the second Fc domain comprises T366S, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, L368A, and Y407V mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations. In some embodiments, the second Fc domain comprises S354C and T366W mutations. In some embodiments, the second Fc domain comprises S354C and N297A mutations. In some embodiments, the second Fc domain comprises T366W and N297A mutations. In some embodiments, the second Fc domain comprises S354C, T366W and N297A mutations.

[0046] In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations and the second Fc domain comprises S354C, T366W and N297A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and N297A mutations and the first Fc domain comprises S354C, T366W and N297A mutations.

[0047] In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, and I253A mutations and the second Fc domain comprises S354C, T366W and I253A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, and I253A mutations and the first Fc domain comprises S354C, T366W and I253A mutations.

[0048] In some embodiments, the first Fc domain comprises Y349C, T366S, L368A, Y407V, N297A, and I253A mutations and the second Fc domain comprises S354C, T366W, N297A and I253A mutations. In some embodiments, the second Fc domain comprises Y349C, T366S, L368A, Y407V, N297A, I253A mutations and the first Fc domain comprises S354C, T366W, N297A and I253A mutations. [0049] In some embodiments, the first Fc polypeptide comprises a first CH3 domain comprising a modification that reduces or eliminates binding to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain comprising a modification that reduces or eliminates binding to Protein A. In some embodiments, the first Fc polypeptide comprises a first CH3 domain that binds to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain that binds to Protein A.

[0050] In some embodiments, the first Fc polypeptide comprises a first CH3 domain comprising a modification that reduces or eliminates binding to Protein A and the second Fc polypeptide comprises a second CH3 domain that binds to Protein A. In some embodiments, the second Fc polypeptide comprises a second CH3 domain comprising a modification that reduces or eliminates binding to Protein A and the first Fc polypeptide comprises a first CH3 domain that binds to Protein A.

[0051] In some embodiments, the CH3 domain that binds to Protein A is a human IgGl sequence. In some embodiments, the CH3 domain that binds to Protein A is a human IgG2 sequence. In some embodiments, the CH3 domain that binds to Protein A is a human IgG4 sequence.

[0052] In some embodiments, the first CH3 domain is a human IgGl sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgGl sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgGl sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgGl sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgGl sequence comprising an Y436F modification. In some embodiments, the first CH3 domain is a human IgGl sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0053] In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising an Y436F modification. In some embodiments, the first CH3 domain is a human IgG2 sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0054] In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising an Y436F modification. In some embodiments, the first CH3 domain is a human IgG3 sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0055] In some embodiments, the second CH3 domain is a human IgGl sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgGl sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgGl sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgGl sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgGl sequence comprising an Y436F modification. In some embodiments, the second CH3 domain is a human IgGl sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0056] In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising an Y436F modification. In some embodiments, the second CH3 domain is a human IgG2 sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0057] In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a modification at position H435 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a modification at position Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a modifications at positions H435 and Y436 according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising a H435R modification according to Kabat numbering. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising an Y436F modification. In some embodiments, the second CH3 domain is a human IgG3 sequence comprising modifications H435R and Y436F according to Kabat numbering.

[0058] In some embodiments, the first Fc domain comprises SEQ ID NO: 85. In some embodiments, the first Fc domain comprises SEQ ID NO: 80. In some embodiments, the second Fc domain comprises SEQ ID NO: 85. In some embodiments, the second Fc domain comprises SEQ ID NO: 80. In some embodiments, the first Fc domain comprises SEQ ID NO: 85 and the second Fc domain comprises SEQ ID NO: 80. In some embodiments, the second Fc domain comprises SEQ ID NO: 85 and the first Fc domain comprises SEQ ID NO: 80.

[0059] In some embodiments, the first CH3 domain comprises a human IgG3 sequence. In some embodiments, the second CH3 domain comprises a human IgG3 sequence.

[0060] In some embodiments, the first Fc domain comprises SEQ ID NO: 84. In some embodiments, the first Fc domain comprises SEQ ID NO: 80. In some embodiments, the second Fc domain comprises SEQ ID NO: 84. In some embodiments, the second Fc domain comprises SEQ ID NO: 80. In some embodiments, the first Fc domain comprises SEQ ID NO: 84 and the second Fc domain comprises SEQ ID NO: 80. In some embodiments, the second Fc domain comprises SEQ ID NO: 84 and the first Fc domain comprises SEQ ID NO: 80.

[0061] In some embodiments, the first antigen binding domain comprises VH, and the second antigen binding domain comprises a VL. In some embodiments, the first antigen binding domain comprises three HCDRs and the second antigen binding domain comprises three LCDRs.

[0062] In some embodiments, the targeting moiety is linked to the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety is linked to the Fc domain through both of the first and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to N-terminus of the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety is linked to N- terminus of the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety is linked to N-terminus of the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety is linked to N-terminus of the Fc domain through both of the first and the second Fc polypeptide. In some embodiments, the targeting moiety is linked to C-terminus of the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety is linked to C-terminus of the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety is linked to C-terminus of the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety is linked to C-terminus of the Fc domain through both of the first and the second Fc polypeptide.

[0063] In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to the Fc domain through both of the first and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to N-terminus of the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to N-terminus of the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to N-terminus of the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to N-terminus of the Fc domain through both of the first and the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to C-terminus of the Fc domain through one or both of the first and second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to C-terminus of the Fc domain through the first Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to C-terminus of the Fc domain through the second Fc polypeptide. In some embodiments, the targeting moiety comprises a variable heavy region (VH) and is linked to C-terminus of the Fc domain through both of the first and the second Fc polypeptide.

[0064] In some embodiments, a cytokine is linked to a first Fc polypeptide through a non-cleavable linker. In some embodiments, the N-terminus of a cytokine is linked to the C-terminus of a first Fc polypeptide through a non-cleavable linker. In some embodiments, the C-terminus of a cytokine is linked to the N-terminus of a first Fc polypeptide through a non-cleavable linker. In some embodiments, a cytokine is linked to a first Fc polypeptide through a cleavable linker. In some embodiments, the N-terminus of a cytokine is linked to the C-terminus of a first Fc polypeptide through a cleavable linker. In some embodiments, the C-terminus of a cytokine is linked to the N-terminus of a first Fc polypeptide through a cleavable linker.

[0065] In some embodiments, a masking moiety is linked to a second Fc polypeptide through a non-cleavable linker. In some embodiments, the N-terminus a masking moiety is linked to the C-terminus of a second Fc polypeptide through a non- cleavable linker. In some embodiments, the C-terminus a masking moiety is linked to the N-terminus of a second Fc polypeptide through a non-cleavable linker. In some embodiments, a masking moiety is linked to a second Fc polypeptide through a cleavable linker. In some embodiments, the N-terminus a masking moiety is linked to the C- terminus of a second Fc polypeptide through a cleavable linker. In some embodiments, the C-terminus a masking moiety is linked to the N-terminus of a second Fc polypeptide through a cleavable linker.

[0066] In some embodiments, the C-terminus of the first antigen binding domain is linked to the N-terminus of the first Fc polypeptide. In some embodiments, the C- terminus of the first Fc polypeptide is linked to the N-terminus of the cytokine or a fragment thereof. In some embodiments, the C-terminus of the second antigen binding domain is linked to the N-terminus of the second Fc polypeptide. In some embodiments, the C-terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety.

[0067] In some embodiments, a targeting moiety comprises an agent that specifically binds to a target. In some embodiments, a targeting moiety comprises a peptide that specifically binds to a target. In some embodiments, a targeting moiety comprises a polypeptide that specifically binds to a target. In some embodiments, a targeting moiety comprises a polypeptide that specifically binds to a target. In some embodiments, a targeting moiety comprises a protein that specifically binds to a target. In some embodiments, a targeting moiety comprises a nucleic acid that specifically binds to a target.

[0068] In some embodiments, a targeting moiety comprises a Fab, a single chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibodies (bsFab), a singledomain antibody-linked Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, a targeting moiety comprises a Fab. In some embodiments, a targeting moiety comprises a single chain Fv (scFv). In some embodiments, a targeting moiety comprises a single domain antibody (VHH). In some embodiments, a targeting moiety comprises one or more CDRs. In some embodiments, a targeting moiety comprises a variable heavy chain (VH). In some embodiments, a targeting moiety comprises a variable light chain (VL). In some embodiments, a targeting moiety comprises a Fab-like bispecific antibodies (bsFab). In some embodiments, a targeting moiety comprises a single-domain antibody-linked Fab (s-Fab). In some embodiments, a targeting moiety comprises an antibody or a fragment thereof.

[0069] In some embodiments, the targeting moiety comprises an antigen-binding moiety, wherein the antigen is expressed on an immune cell. In some embodiments, the targeting moiety specifically binds PD-1. In some embodiments, the targeting moiety specifically binds PD-L1. In some embodiments, the targeting moiety specifically binds PD-L2. In some embodiments, the targeting moiety specifically binds CTLA- 4. In some embodiments, the targeting moiety specifically binds TIGIT. In some embodiments, the targeting moiety specifically binds TIM-3. In some embodiments, the targeting moiety specifically binds LAG-3. In some embodiments, the targeting moiety specifically binds CD25. In some embodiments, the targeting moiety specifically binds CD 16a. In some embodiments, the targeting moiety specifically binds CD 16b. In some embodiments, the targeting moiety specifically binds NKG2D. In some embodiments, the targeting moiety specifically binds NKP44. In some embodiments, the targeting moiety specifically binds NKP3. In some embodiments, the targeting moiety specifically binds CD 19. In some embodiments, the targeting moiety specifically binds CD20. In some embodiments, the targeting moiety specifically binds CD30. In some embodiments, the targeting moiety specifically binds CD38. In some embodiments, the targeting moiety specifically binds BMCA. In some embodiments, the targeting moiety specifically binds HER2. In some embodiments, the targeting moiety specifically binds HER3. In some embodiments, the targeting moiety specifically binds DLL3. In some embodiments, the targeting moiety specifically binds DLL4. In some embodiments, the targeting moiety specifically binds EGFR. In some embodiments, the targeting moiety specifically binds GPC3. In some embodiments, the targeting moiety specifically binds c-MET. In some embodiments, the targeting moiety specifically binds VEGF-R1. In some embodiments, the targeting moiety specifically binds VEGF-R2. In some embodiments, the targeting moiety specifically binds 0X40. In some embodiments, the targeting moiety specifically binds DR5. In some embodiments, the targeting moiety specifically binds ICOS. In some embodiments, the targeting moiety specifically binds GITR. In some embodiments, the targeting moiety specifically binds CD73. In some embodiments, the targeting moiety specifically binds CD39. In some embodiments, the targeting moiety specifically binds CD25. In some embodiments, the targeting moiety specifically binds CD 16a. In some embodiments, the targeting moiety specifically binds CD8. In some embodiments, the targeting moiety specifically binds KLRC1. In some embodiments, the targeting moiety specifically binds KLRD1. In some embodiments, the targeting moiety specifically binds KLRB1. In some embodiments, the targeting moiety specifically binds CD40. In some embodiments, the targeting moiety specifically binds CD137. In some embodiments, the targeting moiety specifically binds CD28.

[0070] In one aspect, the present invention provides, among other things, a nucleic acid encoding the engineered CD 122 or the masked cytokine of the present invention. In one aspect, the present invention provides, among other things, a vector comprising a nucleic acid encoding engineered CD 122 or the masked cytokine of the present invention. In one aspect, the present invention provides, among other things, a host cell comprising a nucleic acid encoding engineered CD 122 or the masked cytokine of the present invention.

[0071] In one aspect, the present invention provides, among other things, a method of producing engineered CD 122 or the masked cytokine comprising culturing the host cell comprising a nucleic acid encoding engineered CD122 or the masked cytokine of the present invention under a condition that produces the engineered CD 122 or the masked cytokine.

[0072] In one aspect, the present invention provides, among other things, a composition comprising the engineered CD122 or the masked cytokine of the present invention.

[0073] In one aspect, the present invention provides, among other things, a pharmaceutical composition comprising the engineered CD122 or the masked cytokine of the present invention and a pharmaceutically acceptable carrier.

[0074] In one aspect, the present invention provides, among other things, a kit comprising the engineered CD122 or the masked cytokine of the present invention.

[0075] In one aspect, the present invention provides, among other things, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the engineered CD 122 or the masked cytokine of the present invention. [0076] In one aspect, the present invention provides, among other things, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising the engineered CD 122 or the masked cytokine of the present invention.

[0077] In one aspect, the present invention provides, among other things, a method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising the engineered CD122 or the masked cytokine of the present invention.

[0078] In one aspect, the present invention provides, among other things, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the engineered CD 122 or the masked cytokine of the present invention.

[0079] In one aspect, the present invention provides, among other things, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a composition comprising the engineered CD 122 or the masked cytokine of the present invention.

[0080] In one aspect, the present invention provides, among other things, a method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising engineered CD122 or the masked cytokine of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The drawings are for illustration purposes only, not for limitation.

[0082] FIG. 1A illustrates exemplary schematics of a masked cytokine comprising a wild-type or an engineered CD122 masking moiety (the engineered CD122 is indicated with a star (*)). FIG. IB illustrates an exemplary schematics of a masked IL-2 cytokine comprising an engineered CD122 masking moiety connected with a cleavable linker. FIG. IB also discloses the amino acid sequence of the cleavable linker. FIG. 1C lists several engineered CD122 or CD122 mutants along with their corresponding mutations. [0083] FIG. 2A illustrates the melting temperatures (Tm) of some selected engineered masked IL-2 cytokines measured by Differential Scanning Calorimetry (DSC) in PBS at pH 7.4. FIG. 2B illustrates DSF and Static Light Scattering (SLS) traces for engineered masked IL-2 cytokine constructs MCI and MC6. The top traces illustrate the fluorescence ratio for the engineered masked IL-2 cytokine constructs MCI and MC6 as a function of temperature. The bottom traces illustrate the intensity of scattered light for the engineered masked IL-2 cytokine constructs MCI and MC6 as a function of temperature. FIG. 2C illustrates the accelerated stability of four selected engineered masked IL-2 cytokine constructs measured at day 0, day 3, day 7, and day 14 by a stress tolerance assay.

[0084] FIG. 3 illustrates exemplary schematics of the Fc domains used in the masked IL-2 cytokines of the present invention. The first Fc polypeptide comprises the CH2 and CH3 domains derived from IgGl. The second chain of the “chimeric Fc” comprise a CH2 domain derived from the IgGl, and a CH3 domain derived from IgG3. The second chain of the “CH3 mutant” comprises CH2 and CH3 domains derived from IgGl with “RF mutations” (H435R and Y436F as indicated with a two stars (**) in the CH3 domain.

[0085] FIG. 4A illustrates a series of exemplary SDS-PAGE analysis of purified masked IL-2 cytokines comprising either the native Fc, CH3 mutant, or chimeric Fc domain. Each masked IL-2 cytokine (also referred as masked IL-2 cytokine construct) was purified by Protein A chromatography and size exclusion chromatography (SEC) at different ratios of the first and second Fc polypeptides. FIG. 4B illustrates a series of exemplary EC50 (i.e., the concentration or dose effective in producing 50% of the maximal response) graphs of purified masked IL-2 cytokines comprising either the native Fc, CH3 mutant, or chimeric Fc domain. EC50 values were determined in a cell -based reporter assay (HEK Blue IL-2 assay) which measures % calculated active cytokine.

[0086] FIG. 5 illustrates exemplary schematics of the targeted IL-2 cytokine (TC) molecules, and their purities as determined by SEC, non-reducing capillary electrophoresis SDS (CE-SDS), and reducing CE-SDS. The TC molecules tested in this experiment all comprise bivalent Fabs (which bind to PD-1), a first Fc polypeptide with “hole mutations” (Y349C; T366S; L368A; and Y407V), and a second Fc polypeptide with “knob mutations” (S354C and T366W) fused to an IL-2 cytokine. TCI is “unmasked,” that is TCI is without a masking moiety that binds to IL-2. TC2, TC3, and TC4 comprise CD122 as a masking moiety that is fused to the first Fc polypeptide. TC3 and TC4 have a cleavable linker comprising MPYDLYHP and VPLSLYSG cleavage peptides, respectively, between the first Fc polypeptide and the masking moiety.

[0087] FIG. 6A illustrates exemplary schematics of different targeted IL-2 cytokines of the present invention. The targeted cytokines can be in a bivalent targeting format (Format A), comprising a bivalent Fabs that bind to a specific antigen or in a monovalent targeting format (Formats Bl, B2, and C) comprising a monovalent Fab. FIG. 6B illustrates a series of exemplary EC50 graphs of the purified targeted IL-2 cytokines shown in FIG. 6A. EC50 values were determined in in a cell-based reporter assay (HEK Blue IL-2 assay) which measures % calculated active cytokine.

[0088] FIG. 7 is an exemplary graph illustrating binding of PD-1 targeting cytokines on CD8+ T cells within CD3 activated PBMCs by frequency anti-hIgG+.

DEFINITIONS

[0089] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0090] It is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0091] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an IL-2 polypeptide” optionally includes a combination of two or more such polypeptides, and the like. [0092] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0093] It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of aspects and embodiments.

[0094] As used herein, the term “and/or” refers to any one of the items, any combination of the items, or all of the items with which the term is associated. For instance, the phrase “A, B, and/or C” is intended to encompass each of the following embodiments: 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 and B or C; B and A or C; C and A or B; A (alone); B (alone); and C (alone).

[0095] The term “antibody” includes polyclonal antibodies, monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules, as well as antibody fragments (e.g., Fab, F(ab’)2, and Fv). The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0096] The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which comprise a heavy chain variable (VH) domain connected to a light chain variable (VL) domain in the same polypeptide chain (VH-VL).

[0097] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. An IgM antibody consists of 5 of the basic heterotetramer units along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of a and y chains and four CH domains for p and s isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

[0098] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, having heavy chains designated a, 8, e, y and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in the CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. IgGl antibodies can exist in multiple polymorphic variants termed allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7) any of which are suitable for use in the invention. Common allotypic variants in human populations are those designated by the letters a,f,n,z.

[0099] An “isolated” antibody is one that has been identified, separated and/or recovered from a component of its production environment (e.g., naturally or recombinantly). In some embodiments, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non- proteinaceous solutes. In some embodiments, the polypeptide is purified: (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody’s natural environment will not be present. Ordinarily, however, an isolated polypeptide or antibody is prepared by at least one purification step.

[0100] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. In some embodiments, monoclonal antibodies have a C- terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the C-terminus of heavy chain and/or light chain. In some embodiments, the C-terminal cleavage removes a C-terminal lysine from the heavy chain. In some embodiments, monoclonal antibodies have an N-terminal cleavage at the heavy chain and/or light chain. For example, 1, 2, 3, 4, or 5 amino acid residues are cleaved at the N-terminus of heavy chain and/or light chain. In some embodiments truncated forms of monoclonal antibodies can be made by recombinant techniques. In some embodiments, monoclonal antibodies are highly specific, being directed against a single antigenic site. In some embodiments, monoclonal antibodies are highly specific, being directed against multiple antigenic sites (such as a bispecific antibody or a multispecific antibody). The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including, for example, the hybridoma method, recombinant DNA methods, phage-display technologies, and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0101] The terms “full-length antibody,” “intact antibody” or “whole antibody” are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antibody fragment. Specifically, whole antibodies include those with heavy and light chains including an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, the intact antibody may have one or more effector functions.

[0102] An “antibody fragment” comprises a portion of an intact antibody, such as the antigen binding region and/or the variable region of the intact antibody, and/or the constant region of the intact antibody. Examples of an antibody fragment include the Fc region of the antibody, a portion of the Fc region, or a portion of the antibody comprising the Fc region. Examples of antigen-binding antibody fragments include domain antibodies (dAbs), Fab, Fab’, F(ab’)2 and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et ah, Protein Eng. 8(10): 1057-1062 [1995]); singlechain antibody molecules, and multispecific antibodies formed from antibody fragments, a single chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibodies (bsFab), a single-domain antibody -linked Fab (s-Fab), and a combination thereof. Single heavy chain antibodies or single light chain antibodies can be engineered, or in the case of the heavy chain, can be isolated from camelids, shark, libraries or mice engineered to produce single heavy chain molecules.

[0103] Papain digestion of antibodies produced two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab’)2 fragment which roughly corresponds to two disulfide linked Fab fragments having different antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab’-SH is the designation herein for Fab’ in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab’)2 antibody fragments originally were produced as pairs of Fab’ fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy -terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences and glycan in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

[0104] “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative subshtuyions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

[0105] Antibody “effector functions” refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0106] “Binding affinity” as used herein refers to the strength of the non-covalent interactions between a single binding site of a molecule (e.g., a cytokine) and its binding partner (e.g., a cytokine receptor). In some embodiments, the affinity of a binding protein (e.g., a cytokine) can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein.

[0107] An “isolated” nucleic acid molecule encoding the cytokine polypeptides described herein is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the environment in which it was produced. In some embodiments, the isolated nucleic acid is free of association with all components associated with the production environment. The isolated nucleic acid molecules encoding the polypeptides and cytokine polypeptides herein is in a form other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from nucleic acid encoding the polypeptides and cytokine polypeptides herein existing naturally in cells.

[0108] The term “pharmaceutical formulation” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0109] Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™.

[0110] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. An individual is successfully “treated”, for example, if one or more symptoms associated with a disorder (e.g., a neoplastic disease) are mitigated or eliminated. For example, an individual is successfully “treated” if treatment results in increasing the quality of life of those suffering from a disease, decreasing the dose of other medications required for treating the disease, reducing the frequency of recurrence of the disease, lessening severity of the disease, delaying the development or progression of the disease, and/or prolonging survival of individuals.

[OHl] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0112] As used herein, the term “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to, susceptible to a disorder, or at risk of developing a disorder, but has not yet been diagnosed with the disorder. In some embodiments, targeted cytokines described herein are used to delay development of a disorder.

[0113] As used herein, an individual “at risk” of developing a disorder may or may not have detectable disease or symptoms of disease, and may or may not have displayed detectable disease or symptoms of disease prior to the treatment methods described herein. “At risk” denotes that an individual has one or more risk factors, which are measurable parameters that correlate with development of the disease, as known in the art. An individual having one or more of these risk factors has a higher probability of developing the disorder than an individual without one or more of these risk factors.

[0114] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired or indicated effect, including a therapeutic or prophylactic result.

[0115] An effective amount can be provided in one or more administrations. A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disorder. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the targeted cytokine are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at the earlier stage of disease, the prophylactically effective amount can be less than the therapeutically effective amount.

[0116] Chronic” administration refers to administration of the medicament(s) in a continuous as opposed to acute mode, so as to main the initial therapeutic effect (activity) for an extended period of time. “Intermittent” administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

[0117] As used herein, an “individual” or a “subject” is a mammal. A “mammal” for purposes of treatment includes humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some embodiments, the individual or subject is human

[0118] Amino acid: As used herein, term “amino acid,” in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. “Standard amino acid” refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, “synthetic amino acid” encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or aminoterminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide’s circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term “amino acid” is used interchangeably with “amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0119] Animal: As used herein, the term “animal” refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.

[0120] Biologically active: As used herein, the phrase “biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.

[0121] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, delivery of mRNA encompasses situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into patient’s circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery).

[0122] Dosing interval: As used herein dosing interval in the context of a method for treating a disease is the frequency of administering a therapeutic composition in a subject (mammal) in need thereof, for example an mRNA composition, at an effective dose of the mRNA, such that one or more symptoms associated with the disease is reduced; or one or more biomarkers associated with the disease is reduced, at least for the period of the dosing interval. Dosing frequency and dosing interval may be used interchangeably in the current disclosure.

[0123] Expression: As used herein, “expression” of a nucleic acid sequence refers to translation of an mRNA into a polypeptide, assemble multiple polypeptides into an intact protein (e.g., enzyme) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., enzyme). In this application, the terms “expression” and “production,” and grammatical equivalent, are used inter-changeably.

[0124] Effective dose: As used herein, an effective dose is a dose of the mRNA in the pharmaceutical composition which when administered to the subject in need thereof, hereby a mammalian subject, according to the methods of the invention, is effective to bring about an expected outcome in the subject, for example reduce a symptom associated with the disease.

[0125] Improve, increase, or reduce: As used herein, the terms “improve,” “increase” or “reduce,” or grammatical equivalents, indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[0126] In Vitro'. As used herein, the term “zzz vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0127] In Vivo'. As used herein, the term “/// vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems). [0128] Patient: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient is a human. A human includes pre- and post-natal forms.

[0129] Pharmaceutically acceptable: The term “pharmaceutically acceptable” as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0130] Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(Cl-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium. quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate. Further pharmaceutically acceptable salts include salts formed from the quarternization of an amine using an appropriate electrophile, e.g., an alkyl halide, to form a quartemized alkylated amino salt.

[0131] Subject: As used herein, the term “subject” refers to a human or any nonhuman animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A human includes pre- and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.

[0132] Substantially: As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0133] Target tissues: As used herein, the term “target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.

[0134] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. [0135] Treating: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[0136] Various aspects of the invention are described in detail in the following sections. The use of sections is not meant to limit the invention. Each section can apply to any aspect of the invention. In this application, the use of “or” means “and/or” unless stated otherwise.

DETAILED DESCRIPTION

CD122 Masking Moiety

[0137] Provided herein is a masking moiety for use in a masked cytokine or in a targeted cytokine. It will be understood that the masking moiety is cleaved from the masked or targeted cytokine to form the cleavage product thereof. The masking moiety masks the IL-2 cytokine or functional fragment thereof in the masked or targeted cytokine thereby reducing or preventing binding of the IL-cytokine or functional fragment thereof to its cognate receptor. In some embodiments, the masking moiety reduces or prevents binding of the IL-2 cytokine or functional fragment thereof to IL-2Ra (CD25). In some embodiments, the masking moiety as provided herein refers to a moiety capable of binding to, or otherwise exhibiting an affinity for the IL-2 cytokine or functional fragment thereof, such as an anti-IL-2 antibody or IL-2 cognate receptor protein. In some embodiments, the masking moiety as provided herein refers to a moiety capable of binding to, or otherwise exhibiting an affinity for the IL- 15 cytokine or functional fragment thereof. Methods for determining the extent of binding of a protein (e.g., cytokine) to a cognate protein (e.g., cytokine receptor) are well known in the art.

[0138] In some embodiments, the masking moiety comprises an IL-2 cytokine receptor, or a subunit or functional fragment thereof. [0139] In some embodiments, the masking moiety comprises IL-2RP (also referred to as CD122) or a fragment, portion, or variant thereof that retains or otherwise demonstrates an affinity to IL-2. In some embodiments, CD122 masking moiety is the wild-type CD122. In some embodiments, CD122 masking moiety is an engineered CD122.

[0140] The present invention provides an engineered CD 122 which has an improved stability. Exemplary wild-type CD122 and engineered CD122 (indicated with a star (*)) masking moi eties capable of binding to a cytokine are shown in FIG. 1A. An exemplary engineered CD 122 masking moiety (indicated with a star (*)) capable of binding to IL-2 cytokine is shown in FIG. IB. The engineered CD122 comprises the amino acid sequence of the wild-type CD122 having specific mutations. FIG. 1C lists some of the exemplary mutations engineered to the wild-type CD 122 to produce several engineered CD 122 or CD 122 mutants.

[0141] In some embodiments, the engineered CD122 comprises mutations selected from a) C122S and C168S, b) Cl 22V and Cl 68V, c) Cl 22 A and Cl 68V, d) Cl 68V, e) Cl 22V and Cl 68 A, f) C122A and N123C, g) C122V and N123C, h) C122A and C168A, i) VI 17C, N123Q and C168A, j) N123Q, C168A, and L169C, k) L106C, C122A, C168A and S195C l) L106C, C122A, C168A and V184C m) C122A, C168A, V184C, and S195C, n) C122A, C168A, Q177C, and R204C, o) L106C, C122V, C168V, and S195C or p) F8C, A94C, C122V, and Cl 68V. [0142] In some embodiments, the masking moiety comprises the amino acid sequence of SEQ ID NO: 15.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 15)

[0143] In some embodiments, the masking moiety comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 15 with one to four amino acid substitutions. In some embodiments, the masking moiety comprises an amino acid sequence having the amino acid sequence of SEQ ID NO: 15 with one or two amino acid substitutions.

[0144] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation at amino acid position Cl 22 as compared to IL-2RP of SEQ ID NO: 15.

[0145] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation C122S at amino acid position 122 as compared to IL-2RP of SEQ ID NO: 15.

[0146] In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 15 with a C122 mutation.

[0147] In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 15 with a C122S mutation.

[0148] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation at amino acid position Cl 68 as compared to IL-2RP of SEQ ID NO: 15.

[0149] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation C168S at amino acid position 168 as compared to IL-2RP of SEQ ID NO: 15. [0150] In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 15 with a C168 mutation.

[0151] In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 15 with a C168S mutation.

[0152] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation at amino acid positions Cl 22 and Cl 68 as compared to IL-2RP of SEQ ID NO: 15.

[0153] In some embodiments, the IL-2RP or a fragment, portion or variant thereof has mutation C122S and C168S as compared to IL-2RP of SEQ ID NO: 15.

[0154] In some embodiments, the masking moiety comprises an amino acid sequence of SEQ ID NO: 16.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRSNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWISLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 16)

[0155] In some embodiments, the masking moiety comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 16. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 16.

[0156] In some embodiments, a masking moiety has a mutation at amino acid positions F8 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation F8C as compared to IL-2RP of SEQ ID NO: 15.

[0157] In some embodiments, a masking moiety has a mutation at amino acid positions A94 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation A94C as compared to IL-2RP of SEQ ID NO: 15.

[0158] In some embodiments, a masking moiety has a mutation at amino acid positions LI 06 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation L106C as compared to IL-2RP of SEQ ID NO: 15.

[0159] In some embodiments, a masking moiety has a mutation at amino acid positions VI 17 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation VI 17C as compared to IL-2RP of SEQ ID NO: 15.

[0160] In some embodiments, a masking moiety has a mutation at amino acid positions Cl 22 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation C122S as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation C122V as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation Cl 22 A as compared to IL-2Rp of SEQ ID NO: 15.

[0161] In some embodiments, a masking moiety has a mutation at amino acid positions N123 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation N123 C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation N123Q as compared to IL-2RP of SEQ ID NO: 15.

[0162] In some embodiments, a masking moiety has a mutation at amino acid positions Cl 68 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation C168S as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation Cl 68V as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation Cl 68 A as compared to IL-2Rp of SEQ ID NO: 15.

[0163] In some embodiments, a masking moiety has a mutation at amino acid positions LI 69 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation L169C as compared to IL-2RP of SEQ ID NO: 15.

[0164] In some embodiments, a masking moiety has a mutation at amino acid positions Q177 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation Q177C as compared to IL-2RP of SEQ ID NO: 15.

[0165] In some embodiments, a masking moiety has a mutation at amino acid positions VI 84 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation V184C as compared to IL-2RP of SEQ ID NO: 15.

[0166] In some embodiments, a masking moiety has a mutation at amino acid positions SI 95 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation S195C as compared to IL-2RP of SEQ ID NO: 15.

[0167] In some embodiments, a masking moiety has a mutation at amino acid positions R204 as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety has a mutation R204C as compared to IL-2RP of SEQ ID NO: 15.

[0168] In some embodiments, a masking moiety has mutations C122V/C168V as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 17.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 17)

[0169] In some embodiments, a masking moiety has mutations C122A/C168V as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 18. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID

NO: 18)

[0170] In some embodiments, a masking moiety has a mutations Cl 68V as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 19.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID

NO: 19)

[0171] In some embodiments, a masking moiety has mutations C122V/C168A as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 20.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID

NO: 20)

[0172] In some embodiments, a masking moiety has mutations C122A/N123C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 21.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRACISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID

NO: 21) [0173] In some embodiments, the masking moiety comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 91% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 93% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 94% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 96% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 98% identity to SEQ ID NO: 21. In some embodiments, the masking moiety comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 21.

[0174] In some embodiments, a masking moiety has mutations C122V/N123C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 22.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRVCISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 22)

[0175] In some embodiments, a masking moiety has mutations C122A/C168A as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 23. AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 23)

[0176] In some embodiments, a masking moiety has mutations V117C/N123Q/C168A as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 24.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHCETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 24)

[0177] In some embodiments, a masking moiety has mutations N123Q/C168A/L169C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 25.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRCQISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIACETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 25)

[0178] In some embodiments, a masking moiety has mutations L106C/C122A/C168A/S195C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 26.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 26) [0179] In some embodiments, a masking moiety has mutations

L106C/C122A/C168A/V184C as compared to IL-2Rp of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 27.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTQYEFQVRCKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID

NO: 27)

[0180] In some embodiments, a masking moiety has mutations

C122A/C168A/V184C/S195C as compared to IL-2RP of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 28.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ

EWIALETLTPDTQYEFQVRCKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID NO: 28)

[0181] In some embodiments, a masking moiety has mutations

C122A/C168A/Q177C/R204C as compared to IL-2Rp of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 29.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS

QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMA PISLQVVHVETHRANISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIALETLTPDTCYEFQVRVKPLQGEFTTWSPWSQPLAFCTKPAALGKD (SEQ ID

NO: 29)

[0182] In some embodiments, a masking moiety has mutations

L106C/C122V/C168V/S195C as compared to IL-2Rp of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 30.

AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRCMA PISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQ EWIVLETLTPDTQYEFQVRVKPLQGEFTTWCPWSQPLAFRTKPAALGKD (SEQ ID

NO: 30)

[0183] In some embodiments, a masking moiety has mutations F8C/A94C/C122V/C168V as compared to IL-2Rp of SEQ ID NO: 15. In some embodiments, a masking moiety comprises an amino acid sequence of SEQ ID NO: 31.

AVNGTSQCTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVS QASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMCIQDFKPFENLRLMAP ISLQVVHVETHRVNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQE WIVLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 31)

[0184] In some embodiments, the engineered CD 122 finds to an IL- 15 cytokine or fragments thereof.

Masked Cytokine

[0185] The present invention provides, among other things, a masked cytokine which comprises a cytokine or a variant thereof, an Fc domain, and a masking moiety. The masking moiety of the masked cytokine binds to the cytokine moiety, protects it, and inhibits the biological activity of the cytokine. In an in vivo environment (e.g., at the site of a disease), the masking moiety is cleaved and released from the masked cytokine, thereby activating the function of cytokine in a target of interest. Exemplary masked cytokines of the present invention are shown in FIG. 1A and FIG. IB.

[0186] In some embodiments, a cytokine is linked to an Fc domain via a cleavable or a non-cleavable linker. In some embodiments, a masking moiety is linked to an Fc domain via a cleavable or a non-cleavable linker. In some embodiments, a masking moiety is linked to an Fc polypeptide via a cleavable linker. In some embodiments, a masking moiety is linked to an Fc polypeptide via a non-cleavable linker. In some embodiments, a cytokine or a variant thereof is linked to an Fc polypeptide via a cleavable linker. In some embodiments, a cytokine or a variant thereof is linked to an Fc polypeptide via a non- cleavable linker. The wild-type and engineered CD 122 of the masked cytokines shown in FIG. 1A and FIG. IB are linked to the Fc domain via a cleavable linker. [0187] Engineered masked IL-2 cytokines of the present invention possess improved stability e.g., improved thermal stability. A skilled artisan will know a method available in the art to measure the thermal stability of a masked cytokine or a polypeptide. In some embodiments, the thermal stability of the masked cytokine is determined by Differential Scanning Fluorimetry (DSF), Differential Scanning Calorimetry (DSC), Pulse-Chase Method, Bleach-chase method, Cycloheximide-chase method, Circular Dichroism (CD) Spectroscopy, or Fluorescence-based Activity Assays.

[0188] In some embodiments, the melting temperature, a measure of thermal stability, of the masked cytokines improved by up to 4 °C. In some embodiments, the melting temperature, a measure of thermal stability, of the masked cytokines improved by 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C.

[0189] Engineered masked IL-2 cytokines of the present invention possess improved stability e.g., superior stress tolerance ability. A skilled artisan will know a method available in the art to measure the stress tolerance ability of a masked cytokine or a polypeptide. In some embodiments, the stress tolerance ability of the masked cytokine is determined by Static Light Scattering (SLS).

[0190] In some embodiments, an engineered masked cytokines of the present invention includes a engineered masked IL-15 cytokine.

Cytokine

[0191] The immune system is skilled in communication and designed to respond quickly, specifically and globally to protect an organism against foreign invaders and disease. The cytokine superfamily of proteins is an integral part of the signaling network between cells and is essential in generating and regulating the immune system. These interacting biological signals have remarkable capabilities, such as influencing growth and development, hematopoiesis, lymphocyte recruitment, T cell subset differentiation, and inflammation.

[0192] Cytokines are part of a bigger immune program, e.g., T cell subset differentiation. Mature CD4 and CD8 T cells leave the thymus with a naive phenotype and produce a variety of cytokines. In the periphery, these T cells encounter antigen presenting cells (APCs) displaying either major histocompatibility complex (MHC) class I molecules (present peptides generated in the cytosol to CD8 T cells) or MHC class II molecules (present peptides degraded in intracellular vesicles to CD4 T cells). Following activation, characteristic cytokine and chemokine secretion profiles allow the classification of CD4 T helper (Th) cells into two major subpopulations in mice and humans.3-7Thl cells secrete mainly IL-2, interferon-y (IFN-y) and tumor necrosis factor-P (TNF-P), whereas Th2 cells secrete mainly IL-4, IL-5, IL-6, IL-10 and IL-13. Thl cells support cell-mediated immunity and as a consequence promote inflammation, cytotoxicity and delayed-type hypersensitivity (DTH). Th2 cells support humoral immunity and serve to downregulate the inflammatory actions of Thl cells. This paradigm is a great example of an integrated biological network and is very useful in simplifying our understanding of typical immune responses and those that turn pathogenic. For example, the failure to communicate “self’ can lead to a loss of tolerance to our own antigens and prompt destructive immune responses to self-tissues and autoimmune disease. Autoimmunity, the major focus of this book, is the underlying mechanism of a set of conditions, such as type 1 diabetes mellitus, multiple sclerosis and rheumatoid arthritis. Autoimmune diseases may be caused in part by cytokine- and chemokine-mediated dysregulation of Th cell subset differentiation. The main factors affecting the development of Th subsets, aside from the context in which the antigen and costimulatory signals are presented, are the cytokines and chemokines in the stimulatory milieu. A better understanding of the properties and interactions of the individual cytokines and chemokines that play a role in Th cell activation may lead to more advanced treatments for autoimmune disease.

[0193] In some embodiments, the masked or targeted cytokine of the present invention comprises an IL-2 cytokine or a variant thereof. In some embodiments, the masked or targeted cytokine of the present invention comprises an IL- 15 cytokine or a variant thereof. In some embodiments, the masked or targeted cytokine of the present invention comprises any cytokine or a variant thereof known in the art that has affinity for or capable of binding to the engineered CD122.

Interleukin 2 (IL-2)

[0194] Provided herein is an IL-2 cytokine or functional fragment thereof for use in a masked or targeted cytokine. IL-2 is an interleukin, which is a type of cytokine signalling molecule in the immune system that regulates activities of white blood cells. [0195] In eukaryotic cells, naturally occurring IL-2 is synthesized as a precursor polypeptide of 153 amino acids, which has SEQ ID NO: 12.

MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKL TRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVI VLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 12)

[0196] This is then processed into mature IL-2 by the removal of amino acid residues 1-20. This results in a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153), which has SEQ ID NO: 13.

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKK ATELKH LQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETA TIVEFLNRWITFAQSIISTLT (SEQ ID NO: 13)

[0197] “Functional fragments” of an IL-2 cytokine comprise a portion of a full length cytokine protein which retains or has modified cytokine receptor binding capability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the full length cytokine protein). Cytokine receptor binding capability can be shown, for example, by the capability of a cytokine to bind to the cytokine’s cognate receptor or a component thereof (e.g., one or more chain(s) of a heterotrimeric receptor complex).

[0198] In some embodiments, the IL-2 cytokine or functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or modified variant thereof capable of binding to an interleukin-2 receptor, particularly the IL-2Ra chain. In the context of IL-2 cytokine binding, the target protein could be IL-2R (comprising the IL-2Ra, IL-2RP, and IL-2Ry chains), the IL-2Ra chain, the IL-2RP chain, or the IL-2Ra/p dimeric complex. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises the amino acid sequence of amino acid residues 21-153 of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or functional fragment thereof comprises the amino acid sequence of mature IL-2, SEQ ID NO: 12.

[0199] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 13. Each of the at least one amino acid modifications can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13.

[0200] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 91% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 92% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 93% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 94% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13.

[0201] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 13 that reduces the affinity of the IL-2 peptide or functional fragment thereof for IL-2Ra (CD25). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13, such that one or more of amino acid residues 38, 42, 45, and 62 is an alanine (A). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13, such that amino acid residues 38, 42, 45, and 62 are an alanine (A).

[0202] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises amino acid sequence substitution C125A as compared to the amino acid sequence of SEQ ID NO: 13.

[0203] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 13, such that amino acid residues 38, 42, 45, and 62 are an alanine (A) and amino acid residue 125 is a alanine (A). In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted for alanine in the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid residues R38, F42, Y45, and E62 substituted for alanine (A) and amino acid residue C125 substituted for alanine (A) in the amino acid sequence of SEQ ID NO: 13.

[0204] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 14.

APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKF AMPKK ATELKH LQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET ATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 14)

[0205] In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 80% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 85% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 90% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 92% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 97% identity to SEQ ID NO: 14. In some embodiments, the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 99% identity to SEQ ID NO: 14.

[0206] In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues e.g. residues 1-3 s removed as compared to the amino acid sequence of the mature IL-2 of SEQ ID NO: 12, for the purpose of removing an O- glycosylation site. In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues substituted as compared to the amino acid sequence of the mature IL-2 of SEQ ID NO: 12, for the purpose of removing an O-glycosylation site. In some embodiments, the IL-2 cytokine or functional fragment thereof has one or more amino acid residues inserted, e.g. in the region of residues 1-3, as compared to the amino acid sequence of the mature IL-2 of SEQ ID NO: 12, for the purpose of removing an O-glycosylation site. In some embodiments, the IL-2 cytokine or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

Interleukin 15 (IL-15)

[0207] Provided herein is an IL- 15 cytokine or functional fragment thereof for use in a masked cytokine or in a targeted cytokine or cleavage product thereof. IL-15 is an interleukin, which is a type of cytokine signalling molecule in the immune system that regulates activities of white blood cells.

[0208] In eukaryotic cells, IL- 15 is synthesized as a precursor polypeptide of 162 amino acids (SEQ ID NO: 92), which is then processed into mature IL-15 by the removal of amino acid residues 1-48. This results in a mature form of IL-15 consisting of 114 amino acids (amino acid residues 49-162) that is secreted in a mature, active form (see SEQ ID NO: 93).

[0209] IL- 15 precursor polypeptide (SEQ ID NO: 92):

MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVN VISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQM FINTS

[0210] IL- 15 mature polypeptide (SEQ ID NO: 93):

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVIS LESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV HIVQMFINTS

[0211] The term “IL- 15” or “IL- 15 polypeptide” as used herein refers to any interleukin- 15 (IL- 15) protein, or a functional fragment or variant thereof. The term encompasses any native IL- 15 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., rats and mice). The term encompasses unprocessed IL- 15 (e.g., a full length, precursor form of IL- 15 that consists of amino acid residues 1-162) as well as any form of IL- 15 that results from processing in the cell (e.g., a mature form of IL-15 that consists of amino acid residues 49-162). As such, the term encompasses a protein encoded by the amino acid sequence of SEQ ID NO: 93, as well as sequence variants thereof. The term also encompasses naturally occurring variants of IL- 15. The term also encompasses non-naturally occurring variants of IL- 15, such as truncations, deletions, forms where IL- 15 is linked to another molecule, and variants caused by at least one amino acid change to the amino acid sequence (e.g., by substitution, addition, or deletion). In some aspects, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, or 114 continuous amino acid portion) compared to a naturally occurring IL- 15 polypeptide, such as an IL- 15 polypeptide encoded by the amino acid sequence of SEQ ID NO: 92 or 93. As such, the term “IL- 15” or “IL- 15 polypeptide” includes an IL- 15 protein comprising the amino acid sequence of SEQ ID NO: 92 or 93, including variants thereof, such as variants created by one or more amino acid substitutions to the amino acid sequence of SEQ ID NO: 92 or 93.

[0212] “Functional fragments” of an IL- 15 cytokine comprise a portion of a full length cytokine protein which retains or has modified cytokine receptor binding capability (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the full length cytokine protein). Cytokine receptor binding capability can be shown, for example, by the capability of a cytokine to bind to the cytokine’s cognate receptor or a component thereof (e.g., one or more chain(s) of a heterotrimeric receptor complex).

[0213] In some embodiments, the IL- 15 cytokine or functional fragment thereof is any naturally occurring interleukin-2 (IL-15) protein or modified variant thereof capable of binding to an interleukin-2 receptor, particularly the IL-15Ra chain.

[0214] In some embodiments, the IL-15 cytokine or fragment thereof comprises SEQ ID NO: 93 or a functional fragment thereof.

[0215] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 93.

[0216] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 93. Each of the at least one amino acid modifications can be any amino acid modification, such as a substitution, insertion, or deletion. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least 5 amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 93. [0217] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 93% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 94% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 93.

[0218] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions as compared to the amino acid sequence of SEQ ID NO: 93.

[0219] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53 as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, N112 as compared to the amino acid sequence of SEQ ID NO: 93. [0220] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position D22 as compared to the amino acid sequence of SEQ ID NO: 93.

[0221] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E46 as compared to the amino acid sequence of SEQ ID NO: 93.

[0222] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position E53 as compared to the amino acid sequence of SEQ ID NO: 93.

[0223] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 as compared to the amino acid sequence of SEQ ID NO: 93.

[0224] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 as compared to the amino acid sequence of SEQ ID NO: 93.

[0225] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N112 as compared to the amino acid sequence of SEQ ID NO: 93.

[0226] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions at positions E46 and E53 as compared to the amino acid sequence of SEQ ID NO: 93.

[0227] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N79 as compared to the amino acid sequence of SEQ ID NO: 93.

[0228] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N112 as compared to the amino acid sequence of SEQ ID NO: 93.

[0229] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. [0230] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71, N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 93.

[0231] In some embodiments, the amino acid substitution at position D22 is D22A.

[0232] In some embodiments, the amino acid substitution at position E46 is E46A.

[0233] In some embodiments, the amino acid substitution at position E46 is E46R.

[0234] In some embodiments, the amino acid substitution at position E46 is E46S.

[0235] In some embodiments, the amino acid substitution at position E53 is E53 A.

[0236] In some embodiments, the amino acid substitution at position E53 is E53R.

[0237] In some embodiments, the amino acid substitution at position E53 is E53S.

[0238] In some embodiments, the amino acid substitution at position N71 is N71Q.

[0239] In some embodiments, the amino acid substitution at position N79 is N79Q.

[0240] In some embodiments, the amino acid substitution at position N112 is N112Q.

[0241] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution D22A as compared to the amino acid sequence of SEQ ID NO: 93.

[0242] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 94.

NWVNVISDLKKIEDLIQSMHIAATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 94)

[0243] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 94.

[0244] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution E46A as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 95.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 95)

[0245] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 95.

[0246] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46A and E53 A as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 96. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 96.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLALQVISLASGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 96)

[0247] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46R and E53R as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 97. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLRLQVISLRSGD

ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 97)

[0248] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having amino acid substitutions E46S and E53S as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 98. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLSLQVISLSSGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 98)

[0249] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution E53 A as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 99. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 99.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLASGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 99)

[0250] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 100 and an amino acid sequence of SEQ ID NO: 101. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100 and an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 101.

NWVNVISDLKKIEDLIQS (SEQ ID NO: 100)

KVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELE EKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 101)

[0251] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 102. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 102.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 102)

[0252] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N79Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 103. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 103.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 103)

[0253] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N112Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 104. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 104)

[0254] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q and N79Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO:

105. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 105.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 105)

[0255] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q and N112Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO:

106. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 106.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILAQNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 106) [0256] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N79Q and N112Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 107. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 107.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILANNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 107)

[0257] In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution N71Q, N79Q and N112Q as compared to the amino acid sequence of SEQ ID NO: 93. In some embodiments, the IL-15 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 108. In some embodiments, the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 108.

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGD ASIHDTVENLIILAQNSLSSNGQVTESGCKECEELEEKNIKEFLQSFVHIVQMFIQTS (SEQ ID NO: 108)

[0258] In some embodiments, an additional mutation may be included in any of the sequences above at position N71. In some embodiments, the mutation is N71A, N71R, N71W, N71F, N71P, N71M, N71L, N71T, N71S, or N71Y.

[0259] In some embodiments, an additional mutation may be included in any of the sequences above at position S73. In some embodiments, the mutation is S73A, S73W, S73V, or S73M.

[0260] In some embodiments, an additional mutation may be included in any of the sequences above at one or more of amino acid positions N72, N79, V80, T81, and N112. In some embodiments, one or more additional mutations selected from N72A, N79A, V80A, T81 A and N112R may be included in any of the sequences above.

[0261] In some embodiments, an additional mutation may be included in any of the sequences above at one or more of amino acid positions N72, S73, N79, V80, T81, and N112. In some embodiments, one or more additional mutations N72A, S73A, N79A, V80A, T81 A, and N112 may be included in any of the sequences above.

[0262] In some embodiments, the IL-15 cytokine or functional fragment thereof has one or more amino acid residues e.g. residues 1-3 s removed as compared to the amino acid sequence of the mature IL- 15 of SEQ ID NO: 93, for the purpose of removing an O- glycosylation site. In some embodiments, the IL- 15 cytokine or functional fragment thereof has one or more amino acid residues substituted as compared to the amino acid sequence of the mature IL- 15 of SEQ ID NO: 93, for the purpose of removing an O- glycosylation site. In some embodiments, the IL- 15 cytokine or functional fragment thereof has one or more amino acid residues inserted, e.g. in the region of residues 1-3, as compared to the amino acid sequence of the mature IL- 15 of SEQ ID NO: 93, for the purpose of removing an O-glycosylation site. In some embodiments, the IL-15 cytokine or functional fragment thereof does not have an O-glycosylation site within residues 1-3.

Targeted Cytokine (Targeted Masked Cytokine)

[0263] The present invention provides, among other things, a targeted cytokine which comprises a targeting moiety, a cytokine or a variant thereof, a masking moiety, and an Fc domain. In some embodiments, the cytokine is an IL-2 cytokine. In some other embodiments, the cytokine is an IL-15 cytokine. In some yet another embodiments, the cytokine is any cytokine or a fragment thereof having binding affinity to the masking moiety, e.g., engineered CD122. Targeted cytokines of the present invention become active at the site of disease, and is able to specifically target a cell of interest for effective treatment of cancer without causing undesired side effects.

[0264] In some embodiments, the cytokine is linked to an Fc domain via a cleavable or a non-cleavable linker. In some embodiments, the masking moiety is linked to an Fc domain via a cleavable or a non-cleavable linker. In some embodiments, a targeting moiety is linked to an Fc domain with or without cleavable or a non-cleavable linker.

[0265] Exemplary targeted IL-2 cytokines of the present invention are shown in FIG. 5 and FIG.6. In some embodiments, a masking moiety is linked to an Fc polypeptide via a cleavable linker. In some embodiments, a masking moiety is linked to an Fc polypeptide via a non-cleavable linker. In some embodiments, an IL-2 cytokine or a variant thereof is linked to an Fc polypeptide via a cleavable linker. In some embodiments, an IL-2 cytokine or a variant thereof is linked to an Fc polypeptide via a non-cleavable linker.

Targeted Cytokine Formats

[0266] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V); 2) a second chain comprising a variable heavy region, a heavy constant region with “knob mutations” (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0267] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V); 2) a second chain comprising a variable heavy region, a heavy constant region from IgGl or IgG4 with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F) fused to a cytokine or a variant thereof; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0268] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V) fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy region, a heavy constant region with “knob mutations” (S354C and T366W) fused to a masking moiety, wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0269] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V) fused to a cytokine or a variant thereof; 2) a second chain comprising a variable heavy region, a heavy constant region from IgGl or IgG4 with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F) fused to a masking moiety; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0270] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy region, a heavy constant region with “knob mutations” (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0271] In some embodiments, a targeted cytokine is a bivalent targeting format, comprising 1) a first chain comprising a variable heavy region and an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V) fused to a masking moiety; 2) a second chain comprising a variable heavy region, a heavy constant region from IgGl or IgG4 with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F) fused to a cytokine or a variant thereof; and 3) a third chain comprising a variable light region and immunoglobulin kappa or lambda constant region.

[0272] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a Fab fused to a first Fc polypeptide chain from an IgGl or IgG4 with “hole mutations” (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a second Fc polypeptide chain from IgGl or IgG4 with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F). [0273] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a Fab fused to a first Fc polypeptide chain from an IgGl or IgG4 with “hole mutations” (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a second Fc polypeptide chain with “knob mutations” (S354C and T366W) wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3.

[0274] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a first Fc polypeptide chain from an IgGl or IgG4 with “hole mutations” (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain from IgGl or IgG4 with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F). In some embodiments, a cytokine is fused to a first Fc polypeptide chain. In some embodiments, a cytokine is fused to a second Fc polypeptide chain. In some embodiments, a masking moiety is fused to a first Fc polypeptide chain. In some embodiments, a masking moiety is fused to a second Fc polypeptide chain.

[0275] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a first Fc polypeptide chain from an IgGl or IgG4 with “hole mutations” (Y349C; T366S; L368A; and Y407V); and 2) a second chain comprising a Fab fused to a second Fc polypeptide chain with “knob mutations” (S354C and T366W) wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3. In some embodiments, a cytokine is fused to a second Fc polypeptide chain. In some embodiments, a masking moiety is fused to a first Fc polypeptide chain. In some embodiments, a masking moiety is fused to a second Fc polypeptide chain.

[0276] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a variable light region and IgG kappa or lambda constant region fused to an IgGl or IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V; and 2) a second chain comprising a variable heavy region fused to an IgGl or IgG4 heavy constant region with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F), wherein the C- terminal of the CH3 domain is fused to a cytokine or a variant thereof. In some embodiments, a cytokine is fused to a second Fc polypeptide chain. In some embodiments, a masking moiety is fused to a first Fc polypeptide chain. In some embodiments, a masking moiety is fused to a second Fc polypeptide chain.

[0277] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a variable light region and IgG kappa or lambda constant region fused to an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V; and 2) a second chain comprising a variable heavy region fused to a heavy constant region with “knob mutations” (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3.

[0278] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a variable heavy region fused to an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L368A; and Y407V; and 2) a second chain comprising a variable light region and IgG kappa or lambda constant region fused to an IgGlor IgG4 heavy constant region with “knob mutations” (S354C and T366W) and “RF mutations” (H435R and Y436F), wherein the C-terminal of the CH3 domain is fused to a cytokine or a variant thereof.

[0279] In some embodiments, a targeted cytokine is a monovalent targeting format, comprising 1) a first chain comprising a variable heavy region fused to an IgGlor IgG4 heavy constant region with “hole mutations” (Y349C; T366S; L638A; and Y407V; and 2) a second chain comprising a light variable region and IgG kappa or lambda constant region fused to a heavy constant region with “knob mutations” (S354C and T366W) fused to a cytokine or a variant thereof, wherein the CHI and CH2 domains are from IgGl or IgG4, and the CH3 domain is from IgG3.

Targeting Moiety

[0280] Provided herein is a targeted cytokine that comprises a targeting moiety. In some embodiments, a targeting moiety comprises an antigen-binding moiety that binds to an antigen expressed on the surface of a target cell. [0281] In some embodiments, the targeting moiety comprises an antigen-binding moiety, wherein the antigen is expressed on an immune cell. In some embodiments, the carrier moiety comprises an antigen-binding moiety, wherein the antigen is selected from PD-1, PD-L1, CTLA- 4, TIGIT, TIM-3, LAG-3, 0X40, DR5, ICOS, GITR, CD73, CD39 CD25, CD16a, CD8, KLRC1, KLRD1, KLRB1, CD40, CD137, CD28 and CD16b.

[0282] In some embodiments, a targeting moiety specifically binds PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, 0X40, DR5, ICOS, GITR, NKG2D, KLRC1, KLRD1, KLRB1, NKP44, NKP30, CD 19, CD20, CD30, CD38, BCMA, human epidermal growth factor receptor 2 (HER2), MICA, DLK1, human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGF Rl), vascular endothelial growth factor receptor 2 (VEG FR2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), NAPI2B, folate receptor alpha (FR-a), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD19, CD8, CD20, CD22, CD28, CD33, CD39, CD40, CD56, CD66e, CD70, CD73, CD74, CD79b, CD98, CD 123, CD137, CD 138, CD352, CD47, signal-regulatory protein alpha (SIRPa), Claudin 18.2, Claudin 6, 5T4, fibroblast activation protein alpha (FAPa), fibronectin, the melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cellular adhesion molecule (EPCAM), or combinations thereof.

[0283] In some embodiments, a targeting moiety specifically binds PD-1. In some embodiments, a targeting moiety binds PD-L1.

[0284] In some embodiments, a targeting moiety comprises an agent, a peptide, or a polypeptide that specifically binds to a target.

[0285] In some embodiments, a targeting moiety comprises a Fab, a single chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibodies (bsFab), a singledomain antibody-linked Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, a targeting moiety comprises a Fab. In some embodiments, a targeting moiety comprises a single chain Fv (scFv). In some embodiments, a targeting moiety comprises a single domain antibody (VHH). In some embodiments, a targeting moiety comprises one or more CDRs. In some embodiments, a targeting moiety comprises a variable heavy chain (VH). In some embodiments, a targeting moiety comprises a variable light chain (VL). In some embodiments, a targeting moiety comprises a Fab-like bispecific antibodies (bsFab). In some embodiments, a targeting moiety comprises a single-domain antibody-linked Fab (s-Fab). In some embodiments, a targeting moiety comprises an antibody or a fragment thereof.

[0286] In some embodiments, a targeting moiety comprises a heavy chain variable region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSS (SEQ ID NO: 1).

[0287] In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 1. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 1.

[0288] In some embodiments, a targeting moiety comprises a heavy chain of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSC (SEQ ID NO: 2)

[0289] In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 2. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 2.

[0290] In some embodiments, a targeting moiety comprises a heavy chain of

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH YTQKSLSLSLGK (SEQ ID NO: 3)

[0291] In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 3 In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 3. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 3.

[0292] In some embodiments, a targeting moiety comprises a heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 4). In some embodiments, a targeting moiety comprises a heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 5). In some embodiments, a targeting moiety comprises a heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 6). In some embodiments, a targeting moiety comprises a HCDR1 of SEQ ID NO: 4, a HCDR2 of SEQ ID NO: 5, and a HCDR3 of SEQ ID NO: 6.

[0293] In some embodiments, a targeting moiety comprises a light chain variable region of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLAS YLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTS ENLYFQ (SEQ ID NO: 7).

[0294] In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 90% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 7. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 7.

[0295] In some embodiments, a targeting moiety comprises a light chain of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLAS YLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 8).

[0296] In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 85% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 90% sequence identity SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 92% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 96% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 97% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 98% sequence identity to SEQ ID NO: 8. In some embodiments, a targeting moiety comprises an amino acid sequence having at least about 99% sequence identity to SEQ ID NO: 8. [0297] In some embodiments, a targeting moiety comprises a light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 9). In some embodiments, a targeting moiety comprises a light chain CDR2 sequence of LAS (SEQ ID NO: 10). In some embodiments, a targeting moiety comprises a light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 11). In some embodiments, a targeting moiety comprises a LCDR1 of SEQ ID NO: 9, a LCDR2 of SEQ ID NO: 10, and a LCDR3 of SEQ ID NO: 11.

[0298] In some embodiments, a targeting moiety comprises a HCDR1 of SEQ ID NO: 4, a HCDR2 of SEQ ID NO: 5, a HCDR3 of SEQ ID NO: 6, a LCDR1 of SEQ ID NO: 9, a LCDR2 of SEQ ID NO: 10, and a LCDR3 of SEQ ID NO: 11.

Linkers

[0299] Provided herein are linkers for use in a targeted cytokine or cleavage product thereof. A linker as provided herein refers to a peptide of two more amino acids that is used to link two functional components together in the targeted cytokines described herein.

[0300] The targeted cytokine comprises a first linker and a second linker, where at least the first linker or the second linker comprises a proteolytically cleavable peptide.

[0301] In some embodiments, a first Fc polypeptide is linked to a cytokine or a variant thereof through a first linker. In some embodiments, a second Fc polypeptide is linked to a masking moiety through a second linker. In some embodiments, a first Fc polypeptide is linked to an IL-2 cytokine or a variant thereof through a cleavable linker. In some embodiments, a second Fc polypeptide is linked to a masking moiety such as engineered CD 122 through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-2 cytokine or a variant thereof through a cleavable linker and a second Fc polypeptide is linked to a masking moiety through a non-cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-2 cytokine or a variant thereof through a non-cleavable linker and a second Fc polypeptide is linked to a masking moiety through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-2 cytokine or a variant thereof through a cleavable linker and a second Fc polypeptide is linked to a masking moiety through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-2 cytokine or a variant thereof through a non-cleavable linker and a second Fc polypeptide is linked to a masking moiety through a non-cleavable linker.

[0302] In some embodiments, a first Fc polypeptide is linked to a cytokine or a variant thereof through a first linker. In some embodiments, a second Fc polypeptide is linked to a masking moiety through a second linker. In some embodiments, a first Fc polypeptide is linked to an IL- 15 cytokine or a variant thereof through a cleavable linker. In some embodiments, a second Fc polypeptide is linked to a masking moiety such as engineered CD 122 through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL- 15 cytokine or a variant thereof through a cleavable linker and a second Fc polypeptide is linked to a masking moiety through a non-cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-15 cytokine or a variant thereof through a non-cleavable linker and a second Fc polypeptide is linked to a masking moiety through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-15 cytokine or a variant thereof through a cleavable linker and a second Fc polypeptide is linked to a masking moiety through a cleavable linker. In some embodiments, a first Fc polypeptide is linked to an IL-15 cytokine or a variant thereof through a non-cleavable linker and a second Fc polypeptide is linked to a masking moiety through a non-cleavable linker.

Non-Cleavable Linker

[0303] In some embodiments, the non-cleavable linker is between 2 and 25 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 21 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 18 amino acids in length. In some embodiments, the non-cleavable linker is between 5 and 18 amino acids in length. In some embodiments, the non-cleavable linker is between 3 and 8 amino acids in length. In some embodiments, the non-cleavable linker is between 4 and 6 amino acids in length.

[0304] In some embodiments, the non-cleavable linker is 15 amino acids in length. In some embodiments, the non-cleavable linker is 16 amino acids in length. In some embodiments, the non-cleavable linker is 17 amino acids in length. In some embodiments, the non-cleavable linker is 18 amino acids in length. In some embodiments, the non-cleavable linker is 19 amino acids in length. In some embodiments, the non- cleavable linker is 20 amino acids in length.

[0305] In some embodiments, the non-cleavable linker is rich in amino acid residues G, S and P. In some embodiments, the non-cleavable linker only includes amino acid residue types selected from the group consisting of G, S and P. In some embodiments, the non-cleavable linker includes a ‘GS’ repeat. In some embodiments, the non-cleavable linker includes an N’ terminal ‘P’ residue.

[0306] In some embodiments, the non-cleavable linker comprises an amino acid sequence as shown in SEQ ID NO: 32 (PGSGS).

[0307] In some embodiments, the non-cleavable linker comprises an amino acid sequence as shown in SEQ ID NO: 33 (GGSSPPGGGSSGGGSGP).

[0308] In some embodiments, the non-cleavable linker comprises an amino acid sequence GGS.

Cleavable Linker

[0309] In some embodiments, the cleavable linker is between 2 and 25 amino acids in length. In some embodiments, the cleavable linker is between 3 and 21 amino acids in length. In some embodiments, the cleavable linker is between 3 and 18 amino acids in length. In some embodiments, the cleavable linker is between 5 and 18 amino acids in length. In some embodiments, the cleavable linker is between 3 and 8 amino acids in length. In some embodiments, the cleavable linker is between 4 and 6 amino acids in length.

[0310] In some embodiments, the cleavable linker is 15 amino acids in length. In some embodiments, the cleavable linker is 16 amino acids in length. In some embodiments, the cleavable linker is 17 amino acids in length. In some embodiments, the cleavable linker is 18 amino acids in length. In some embodiments, the cleavable linker is 19 amino acids in length. In some embodiments, the cleavable linker is 20 amino acids in length.

[0311] In some embodiments, the cleavable linker comprises a proteolytically cleavable peptide (CP) flanked on both sides by a spacer domain (SD) as shown in below formula: SD-CP-SD

Cleavable Peptides

[0312] The cleavable linker comprises a cleavable peptide.

[0313] A cleavable peptide is a polypeptide that includes a protease cleavage site, such that the cleavable peptide is proteolytically cleavable. Proteases are enzymes that cleave and hydrolyse the peptide bonds between two specific amino acid residues of target substrate proteins. A “cleavage site” as used herein refers to a recognizable site for cleavage of a portion of the cleavable peptide found in any of the linkers that comprise a cleavable peptide described herein. Thus, a cleavage site may be found in the sequence of a cleavable peptide as described herein. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleaving agent.

[0314] In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. A “tumor-associated protease cleavage site” as provided herein is an amino acid sequence recognized by a protease whose expression is specific or upregulated for a tumor cell or tumor cell environment thereof.

[0315] The tumor cell environment is complex and can comprise multiple different proteases. As such, the precise site at which a given cleavable peptide will be cleaved in the tumor cell environment may vary between tumor types, between patients with the same tumor type and even between cleavage products formed in the same tumor dependent on the specific tumor cell environment. Moreover, even after cleavage, further modification of the initial cleavage product, e.g. by removal of one or two terminal amino acids, may occur by the further action of proteases in the tumor cell environment. A distribution of cleavage products can thus be expected to form in the tumor cell environment of a patient following administration of a single structure of a targeted cytokine as described herein.

[0316] It will be understood that a cleavage site as referred to herein refers to a site between two specific amino acid residues within the cleavable peptide that are a target for a protease known to be associated with a tumor cell environment. In this sense, there may be more than one cleavage site present in a cleavable peptide as described herein where different proteases cleave the cleavable peptide at different cleavage sites. It is also possible that more than one protease may act on the same cleavage site within a cleavable peptide. Discussion of protease cleavage sites can be found in the art.

[0317] Thus, the cleavable peptides disclosed herein may be cleaved by one or more proteases.

[0318] In some embodiments, the cleavable peptide is a substrate for a protease that is co-localized in a region or a tissue expressing the IL-2 cytokine receptor, particularly IL-2Ra.

[0319] In some embodiments, the cleavable peptide is a 5-mer (i.e. peptide 5 amino acids in length), 6-mer (i.e. peptide 6 amino acids in length), 7-mer (i.e. peptide 7 amino acids in length), 8-mer (i.e. peptide 8 amino acids in length), 9-mer (i.e. peptide 9 amino acids in length), 10-mer (i.e. peptide 10 amino acids in length), 11-mer (i.e. peptide 11 amino acids in length), 12-mer (i.e. peptide 12 amino acids in length), 13-mer (i.e. peptide 13 amino acids in length), 14-mer (i.e. peptide 14 amino acids in length), 15-mer (i.e. peptide 15 amino acids in length), 16-mer (i.e. peptide 16 amino acids in length), 17- mer (i.e. peptide 17 amino acids in length), or 18-mer (i.e. peptide 18 amino acids in length).

[0320] In some embodiments, the cleavable peptide is from 5 to 18 amino acids in length. In some embodiments, the cleavable peptide is from 6 to 10 amino acids in length.

[0321] In some embodiments, the cleavable peptide within the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40. In some embodiments, the cleavable peptide within the cleavable linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, and 40.

[0322] In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 34. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 35. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 36. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 37. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 38. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 39. In some embodiments, the cleavable peptide within the cleavable linker comprises SEQ ID NO: 40.

[0323] Purely by way of example, in the above table, * indicates a known or observed protease cleavage site within the cleavable peptide.

[0324] In some embodiments, the cleavable peptide comprises an amino acid sequence of SEQ ID NO: 41 (MPYDLYHPS). In some embodiments, the cleavable peptide may comprise an amino acid sequence of SEQ ID NO: 42 (VPLSLYSG). In some embodiments, the cleavable peptide may comprise an amino acid sequence of SEQ ID NO: 43 (ISSGLLSGRSDQP).

[0325] In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 44. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 45. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 46. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 47. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 48. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 49. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 50. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 51. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 52. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 53. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 54. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 56. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 58. In some embodiments, the cleavable linker comprises an amino acid sequence of SEQ ID NO: 59.

Spacer Domains

[0326] A spacer domain may consist of one or more amino acids. The function of the spacer domains, where present, is to link the proteolytically cleavable peptide (CP) to the other functional components in the constructs described herein.

[0327] It will be understood that spacer domains do not alter the biological interaction of the proteolytically cleavable peptide with proteases in the tumor-cell environment or in non-tumor cell environment. In other words, even in the presence of spacer domains the inventive proteolytically cleavable peptides disclosed herein retain their advantageous tumor specificity. [0328] In some embodiments, the spacer domains flanking the proteolytically cleavable peptide are different.

[0329] In some embodiments, the spacer domains are rich in amino acid residues G, S and P.

[0330] In some embodiments, the spacer domains only includes amino acid residue types selected from the group consisting of G, S and P.

[0331] In some embodiments, the cleavable linker comprises formula 12:

N’ SD1-CP-SD2 C’ (12) where SD1 is a first spacer domain and SD2 is a second spacer domain.

[0332] In some embodiments, the cleavable linker comprises formula 12:

N’ SD1-CP-SD2 C’ (12)

[0333] In some embodiments, the first polypeptide chain comprises formula 7 and the second polypeptide chain comprises formula 13 below:

N’ HLl-non-cleavable Ll-MM C’ (7)

N’ HL2- SD1-CP-SD2 -C C’ (13)

[0334] In some embodiments, the first polypeptide chain comprises formula 14 and the second polypeptide chain comprises formula 10 below:

N’ HL1- SD1-CP-SD2 -MM C’ (14)

N’ HL2- non-cleavable L2-C C’ (10)

[0335] In some embodiments, SD1 consists of a glycine (G).

[0336] In some embodiments, the N-terminus of SD1 is a glycine (G).

[0337] In some embodiments, the first spacer domain (SD1) is between 3 and 10 amino acids in length. In some embodiments, the first spacer domain (SD1) is between 4 and 9 amino acids in length. In some embodiments, the first spacer domain (SD1) is between 3 and 6 amino acids in length. [0338] In some embodiments, SD1 comprises SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72. In some embodiments, SD2 comprises SEQ ID NO: 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 or 72.

[0339] In some embodiments, the SD2 consists of GP.

[0340] In some embodiments, the C-terminus sequence of SD2 is -GP C’.

[0341] In some embodiments, the second spacer domain (SD2) is between 3 and 6 amino acids in length.

[0342] In some embodiments, SD2 comprises SEQ ID NO: 73, 74, or 75.

[0343] In some embodiments, SD2 consists of SEQ ID NO: 73, 74, or 75.

[0344] Exemplary combinations of SD1 and SD2 in a cleavable linker are shown below:

Fc domain

[0345] Provided herein are Fc domains for use in a masked or targeted cytokine or cleavage product thereof. A long half-life in vivo is important for therapeutic proteins. Unfortunately, cytokines that are administered to a subject generally have a short half-life since they are normally cleared rapidly from the subject by mechanisms including clearance by the kidney and endocytic degradation. Thus, in the masked or targeted cytokine provided herein, an Fc domain is linked to the cytokine or a masking moiety for the purpose of extending the half-life of the cytokine in vivo, among other things.

[0346] In some embodiments, an Fc domain comprises a first Fc polypeptide and a second Fc polypeptide.

[0347] An Fc domain or a fragment thereof that is capable of FcRn-mediated recycling, can be reduce or otherwise delay clearance of the targeted cytokine from a subject, thereby prolonging the half-life of the administered targeted cytokine. In some embodiments, the Fc domain or a fragment thereof is any antibody or fragment thereof that is capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., Fc domain or fragment thereof) that is capable of FcRn-mediated recycling.

[0348] The Fc domain or a fragment thereof can be any antibody or fragment thereof. However, in some embodiments, either a first Fc polypeptide or a second Fc polypeptide may does not bind to the FcRn receptor, such as a light chain polypeptide. For example, in some embodiments, a first Fc polypeptide does not directly interact with the FcRn receptor, but the targeted cytokine nonetheless has an extended half-life due to comprising a second Fc polypeptide that is capable of interacting with the FcRn receptor, such as by comprising a heavy chain polypeptide. It is recognized in the art that FcRn- mediated recycling requires binding of the FcRn receptor to the Fc region of the antibody or fragment thereof. For instance, studies have shown that residues 1253, S254, H435, and Y436 (numbering according to the Kabat EU index numbering system) are important for the interaction between the human Fc region and the human FcRn complex. See, e.g., Firan, M., et al., Int. Immunol. 13 (2001) 993-1002; Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbering according to the Kabat EU index numbering system) have also been examined and reported. Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671.

[0349] In some embodiments, the antibody or fragment thereof comprises either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a portion of either a heavy chain polypeptide or a light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises an Fc domain or fragment thereof. In some embodiments, the antibody or fragment thereof comprises a CH2 and CH3 domain or a fragment thereof. In some embodiments, the antibody or fragment thereof comprises the constant domain of the heavy chain polypeptide. In some embodiments, the antibody or fragment thereof comprises the constant domain of the light chain polypeptide. In some embodiments, the antibody or fragment thereof comprises a heavy chain polypeptide or fragment thereof (e.g., an Fc domain or fragment thereof). In some embodiments, the antibody or fragment thereof comprises a light chain polypeptide. [0350] In some embodiments, the first and/ or second Fc domains each contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptides. In some embodiments, the first Fc polypeptide comprises an IgGl Fc domain or fragment thereof including the mutations Y349C; T366S; L368A; and Y407V to form a ‘hole’ in the first half-life extension domain and the second Fc polypeptide comprises an IgGl Fc domain or fragment thereof including the mutations S354C and T366W to form the ‘knob’ in the second half-life extension domain.

[0351] In some embodiments, the first and second Fc polypeptides are each an IgGl, IgG2 or IgG4 Fc domain or fragment thereof. In some embodiments, the first and second Fc polypeptides are each an IgGl Fc domain or fragment thereof. Human IgGl Immunoglobulin heavy constant gamma 1 has the sequence:

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 76)

[0352] In some embodiments, the first and second Fc polypeptides are derived from the sequence for human IgGl Immunoglobulin heavy constant gamma 1 having SEQ ID NO: 76 (the ‘parent sequence’), such that the first and second Fc polypeptides each comprise SEQ ID NO: 76 or fragment thereof, with one or more amino acid modifications.

[0353] In some embodiments, the first and Fc polypeptides each comprise the portion of SEQ ID NO: 76 shown in bold above, optionally with one or more amino acid modifications, i.e.:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG (SEQ ID NO: 77) [0354] In some embodiments, the first and second Fc polypeptides comprise SEQ ID NO: 77 with amino substitutions to promote association of the first and second Fc polypeptides according to the ‘knob into holes’ approach. In some embodiments, the sequence SEQ ID NO: 77 contains mutations Y349C; T366S; L368A; and Y407V (numbered according to the Kabat EU numbering system) to form the ‘hole’ in the first Fc polypeptide and mutations S354C and T366W (numbered according to the Kabat EU numbering system) to form the ‘knob’ in the second Fc polypeptide. These modified sequences have SEQ ID NOs 78 and 79 shown below:

[0355] First Fc polypeptide (Y349C; T366S; L368A; and Y407V) SEQ ID NO 78:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

[0356] Second Fc polypeptide (S354C and T366W) SEQ ID NO 79:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<AL PAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

[0357] In some embodiments, the first and second half-life extension domains each further comprise amino substitution N297A, numbered according to the Kabat EU numbering system:

[0358] First Fc polypeptide (Y349C; T366S; L368A; Y407V and N297A) SEQ ID

NO 80:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG [0359] Second Fc polypeptide (S354C, T366W and N297A) SEQ ID NO 81:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

[0360] In some embodiments, the first and second Fc polypeptides each further comprise the amino substitution 1253 A, numbered according to the Kabat EU numbering system.

[0361] In some embodiments, the first and second Fc polypeptides each further comprise both the amino substitutions N297A and 1253 A, numbered according to the Kabat EU numbering system.

[0362] First Fc polypeptide (Y349C; T366S; L368A; Y407V, N297A and 1253 A)

SEQ ID NO 82:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFN

WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

[0363] Second Fc polypeptide (S354C, T366W, N297A and I253A) SEQ ID NO 83:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMASRTPEVTCVVVDVSHEDPEVKFN

[0364] In some embodiments, the first Fc polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequence of any one of SEQ ID NOs: 77-83. [0365] In some embodiments, the second Fc polypeptide comprises an amino acid sequence having about or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the amino acid sequence of any one of SEQ ID NOs: 77-83.

[0366] In some embodiments, the first Fc polypeptide comprises an amino acid sequence having one or more modifications, such as one or more amino acid substitutions, additions, or deletions, as compared to the amino acid sequence of any one of SEQ ID NOs: 77-83. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having one or more modifications, such as one or more amino acid substitutions, additions, or deletions, as compared to the amino acid sequence of any one of SEQ ID NOs: 77-83. The one or more modifications can be any modifications or alterations described herein, including, in some embodiments, any modifications or alterations disclosed herein that promote heterodimerization of polypeptide chains and/or suppresses homodimerization of polypeptide chains, alter effector function, or enhance effector function.

[0367] In some embodiments, the Fc domain or fragment thereof comprises one or more amino acid substitutions altering effector function. In some embodiments, the halflife extension domain is an IgGl Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297G, N297Q, L234A, L235A, C220S, C226S, C229S, P238S, E233P, L234V, L234F, L235E, P331 S, S267E, L328F, D265 A, and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof and comprises the amino substitution(s): V234A and G237A; H268Q, V309L, A330S, and A331S; and/or V234A, G237A, P238S, H268A, V309L, and A330S, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of V234A, G237A, H268Q, V309L, A330S, A331S, P238S, H268A, and V309L, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG4 Fc domain or fragment thereof and comprises the amino substitution(s): L235A, G237A, and E318A; S228P, L234A, and L235A; H268Q, V309L, A330S, and P331 S; and/or S228P and L235A, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or fragment thereof and comprises one or more amino acid substitutions selected from the group consisting of L235A, G237A, E318A, S228P, L234A, H268Q, V309L, A330S, and P331S, numbered according to the Kabat EU numbering system.

[0368] In some embodiments, the Fc domain or fragment thereof that comprises one or more amino acid substitutions enhancing effector function. In some embodiments, the half-life extension domain is an IgGl Fc domain or fragment thereof and comprises the amino acid substitution(s): S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S, and either S298D or S298V; F243L, R292P, and Y300L; F243L, R292P, Y300L, and P396L; F243L, R292P, Y300L, V305I, and P396L; G236A, S239D, and I332E; K326A and E333A; K326W and E333S; K290E, S298G, and T299A; K290E, S298G, T299A, and K326E; K290N, S298G, and T299A; K290N, S298G, T299A, and K326E; K334V;

L235S, S239D, and K334V; K334V and Q331M, S239D, F243V, E294L, or S298T;

E233L, Q311M, and K334V; L234I, Q311M, and K334V; K334V and S298T, A330M, or A330F; K334V, Q31 IM, and either A330M or A330F; K334V, S298T, and either A330M or A330F; K334V, S239D, and either A330M or S298T; L234Y, Y296W, and K290Y, F243 V, or E294L; Y296W and either L234Y or K290Y; S239D, A330S, and I332E, V264I; F243L and V264I; L328M; I332E; L328M and I332E; V264I and I332E; S239E and I332E; S239Q and I332E; S239E; A330Y; I332D; L328I and I332E; L328Q and I332E; V264T; V240I; V266I; S239D; S239D and I332D; S239D and I332N; S239D and I332Q; S239E and I332D; S239E and I332N; S239E and I332Q; S239N and I332D; S239N and I332E; S239Q and I332D; A330Y and I332E; V264I, A330Y, and I332E;

A330L and I332E; V264I, A330L, and I332E; L234E, L234Y, or L234I; L235D, L235S, L235Y, or L235I; S239T; V240M; V264Y; A330I; N325T; I332E and L328D, L328V, L328T, or L328I; V264I, I332E, and either S239E or S239Q; S239E, V264I, A330Y, and I332E; A330Y, I332E, and either S239D or S239N; A330L, I332E, and either S239D or S239N; V264I, S298A, and I332E; S298A, I332E, and either S239D or S239N; S239D, V264I, and I332E; S239D, V264I, S298A, and I332E; S239D, V264I, A330L, and I332E; S239D, I332E, and A330I; P230A; P230A, E233D, and I332E; E272Y; K274T, K274E, K274R, K274L, or K274Y; F275W; N276L; Y278T; V302I; E318R; S324D, S324I or S324V; K326I or K326T; T335D, T335R, or T335Y; V240I and V266I; S239D, A330Y, I332E, and L234I; S239D, A330Y, I332E, and L235D; S239D, A330Y, I332E, and V240I; S239D, A330Y, I332E, and V264T; and/or S239D, A330Y, I332E, and either K326E or K326T, numbered according to the Kabat EU numbering system. In some embodiments, the Fc domain is an IgGl Fc domain or fragment thereof and comprises one or more amino acid substitution(s) selected from the group consisting of: P230A, E233D, L234E, L234Y, L234I, L235D, L235S, L235Y, L235I, S239D, S239E, S239N, S239Q, S239T, V240I, V240M, F243L, V264I, V264T, V264Y, V266I, E272Y, K274T, K274E, K274R, K274L, K274Y, F275W, N276L, Y278T, V302I, E318R, S324D, S324I, S324V, N325T, K326I, K326T, L328M, L328I, L328Q, L328D, L328V, L328T, A330Y, A330L, A330I, I332D, I332E, I332N, I332Q, T335D, T335R, and T335Y.

[0369] In some embodiments, the Fc domain comprises one or more amino acid substitution(s) that enhance binding of the half-life extension domain to FcRn. In some embodiments, the one or more amino acid substitution(s) increase binding affinity of an Fc-containing polypeptide (e.g., a heavy chain polypeptide or an Fc domain or fragment thereof) to FcRn at acidic pH. In some embodiments, the half-life extension domain comprises one or more amino acid substitution(s) selected from the group consisting of M428F; T250Q and M428F; M252Y, S254T, and T256E; P257I and N434H; D376V and N434H; P257I and Q3111; N434A; N434W; M428F and N434S; V259I and V308F; M252Y, S254T, and T256E; V259I, V308F and M428F; T307Q and N434A; T307Q and N434S; T307Q, E380A, and N434A; V308P and N434A; N434H; and V308P.

Knobs-into-Holes Approach

[0370] One strategy for promoting heterodimerization of two Fc polypeptides is an approach termed the “knobs-into-holes”.

[0371] In some embodiments, the targeted cytokine comprises a first Fc polypeptide and a second Fc polypeptide, each of which comprises a CH3 domain. In some embodiments, the Fc polypeptide comprising a CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or fragment thereof). The CH3 domains of the two Fc polypeptides can be altered by the “knobs-into-holes” technology, which is described in detail with several examples in, e.g., WO 1996/027011; Ridgway, J.B. et al, Protein Eng. (1996) 9(7): 617-621; Merchant, A.M., et al, Nat. Biotechnol.

(1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653-663. Using the knob- into-holes method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two half-life extension domains containing the two altered CH3 domains. This occurs by introducing a bulky residue into the CH3 domain of one of the half-life extension domains, which acts as the “knob.” Then, in order to accommodate the bulky residue, a “hole” is formed in the other half-life extension domain that can accommodate the knob. Either of the altered CH3 domains can be the “knob” while the other can be the “hole.” The introduction of a disulfide bridge further stabilizes the heterodimers (Merchant, A.M., et al, Nat. Biotechnol. (1998) 16(7); Atwell, S., et al, J. Mol. Biol. (1997) 270(1): 26-35) as well as increases yield.

[0372] It has been reported that heterodimerization yields above 97% can be achieved by introducing the S354C and T366W mutations in a heavy chain to create the “knob” and by introducing the Y349C, T366S, L368A, and Y407V mutations in a heavy chain to create the “hole” (numbering of the residues according to the Kabat EU numbering system). Carter et al. (2001), J. Immunol. Methods, 248: 7-15; Klein et al. (2012), MAbs, 4(6): 653-663.

[0373] In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first half-life Fc polypeptide comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system). In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises the amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) that comprises the amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system).

[0374] Additional examples of substitutions that can be made to form knobs and holes include those described in US20140302037A1, the contents of which are herein incorporated by reference. For example, in some embodiments, any of the following amino acid substitutions can be made to a first Fc polypeptide (“first domain”) and a paired second Fc polypeptide (“second domain”) that each contain an Fc domain: (a) Y407T in the first domain and T366Y in the second domain; (b) Y407A in the first domain and T366W in the second domain; (c) F405A in the first domain and T394W in the second domain; (d) F405W in the first domain and T394S in the second domain; (e) Y407T in the first domain and T366Y in the second domain; (f) T366Y and F405A in the first domain and T394W and Y407T in the second domain; (g) T366W and F405W in the first domain and T394S and Y407A in the second domain; (h) F405W and Y407A in the first domain and T366W and T394S in the second domain; or (i) T366W in the first domain and T366S, L368A, and Y407V in the second domain, numbered according to the Kabat EU numbering system.

[0375] In some embodiments, any of the following amino acid substitutions can be made to a first Fc polypeptide (“first domain”) and a paired second Fc polypeptide (“second domain”) that each contain an Fc domain: (a) Y407T in the second domain and T366Y in the first domain; (b) Y407A in the second domain and T366W in the first domain; (c) F405A in the second domain and T394W in the first domain; (d) F405W in the second domain and T394S in the first domain; (e) Y407T in the second domain and T366Y in the first domain; (f) T366Y and F405A in the second domain and T394W and Y407T in the first domain; (g) T366W and F405W in the second domain and T394S and Y407A in the first domain; (h) F405W and Y407A in the second domain and T366W and T394S in the first domain; or (i) T366W in the second domain and T366S, L368A, and Y407V in the first domain, numbered according to the Kabat EU numbering system.

[0376] In embodiments comprising a first Fc polypeptide and a second Fc polypeptide that each comprise an Fc domain, any of the heterodimerizing alterations described herein can be used in the Fc domains to promote heterodimerization of any of the targeted cytokines described herein.

RF mutation or CH 3 domain swap for Heterodimeric Protein Purification

[0377] Two immunoglobulin heavy chains that differ by at least one amino acid allows isolation of the antigen-binding protein based on a differential affinity of an immunoglobulin heavy chain and a modified or mutated immunoglobulin heavy chain toward an affinity reagent. The antigen-binding proteins that have IgG CH2 and CH3 regions with different affinities with respect to Protein A allow rapid isolation by differential binding of the IgG regions to Protein A.

[0378] In one embodiment, a second Fc polypeptide comprises a 95R modification (by IMGT exon numbering; 435R by EU numbering) in the CH3 region. In another embodiment, a second Fc polypeptide further comprises a 96F modification (IMGT; 436F by EU). In some embodiments, a first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgGl or IgG4, and a second Fc polypeptide comprises 95R/96F modifications by IMGT exon numbering. In some embodiments, a first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgGl or IgG4, and a second Fc polypeptide comprises 435R/436F modifications by EU numbering.

[0379] In some embodiments, a first Fc polypeptide comprises wild-type CH2 and

CH3 domains derived from IgGl or IgG4, and a second Fc polypeptide comprises CH3 domain derived from IgG3.

[0380] In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence of SEQ ID NO: 84. SEQ ID NO: 84 comprises “knob mutations” with CH3 domain from IgG3.

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCREELTKNQVSLWCLVKGFYPSDIAVEWESS GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQK SLSLSPGGSPG (SEQ ID NO: 84)

[0381] In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence of SEQ ID NO: 85. SEQ ID NO: 85 comprises “knob mutations” with “RF mutations (435R/436F).

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQ KSLSLSPGGSPG (SEQ ID NO: 85) [0382] In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence of SEQ ID NO: 86.

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQ KSLSLSPG (SEQ ID NO: 86)

[0383] In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 85% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 90% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 91% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 92% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 93% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 94% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 95% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 96% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 97% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 98% identity to SEQ ID NO: 86. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 99% identity to SEQ ID NO: 86.

[0384] In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 85% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 90% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 91% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 92% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 93% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 94% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 95% identity to SEQ ID NO: 0. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 96% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 97% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 98% identity to SEQ ID NO: 80. In some embodiments, a first Fc polypeptide or a second Fc polypeptide comprises amino acid sequence having at least about 99% identity to SEQ ID NO: 80.

Exemplary Targeted Cytokine

[0385] In some embodiments, a targeted IL-2 cytokine of the present invention comprises a first chain comprising SEQ ID NO: 87, a second chain comprising SEQ ID NO: 88, and a third chain comprising SEQ ID NO: 8.

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVI<FNWYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CI<V SNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NRFTQKSLSLSPGGGSSPPGGGSSGGGSGPAPTSSSTKKTQLQLEHLLLDLQMILN GINNYI<NPI<LTAMLTAI<FAMPI<I<ATELI<HLQCLEEALI<PLEEVLNLAQSI<NFHL

RPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ

ID NO: 87)

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

EVI<FNWYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CT<V

SNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWS QDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDI VTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRSNISWEISQAS

HYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQ GEFTTWSPWSQPL AFRTKP AALGKD (SEQ ID NO: 88)

[0386] In some embodiments, a targeted IL-2 cytokine of the present invention comprises a first chain comprising SEQ ID NO: 87, a second chain comprising SEQ ID NO: 89, and a third chain comprising SEQ ID NO: 8.

EVI<FNWYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CT<V

SNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGPPSGSSPMPYDLYHPSGGGAVNGTSQFTCFYNSRANISCVWS QDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDI VTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRACISWEISQAS HYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPL

QGEFTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 89)

[0387] In some embodiments, a targeted IL-2 cytokine of the present invention comprises a first chain comprising SEQ ID NO: 87, a second chain comprising SEQ ID

NO: 90, and a third chain comprising SEQ ID NO: 8.

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN

PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMG FDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

EVI<FNWYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CI<V

SNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGSSGPVPLSLYSGSGGGAVNGTSQFTCFYNSRANISCVWSQD GALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVT LRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRSNISWEISQASHY

FERHLEFEARTLSPGHTWEEAPLLTLKQKQEWISLETLTPDTQYEFQVRVKPLQGE

FTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 90)

[0388] In some embodiments, a targeted IL-2 cytokine of the present invention comprises a first chain comprising SEQ ID NO: 87, a second chain comprising SEQ ID NO: 91, and a third chain comprising SEQ ID NO: 8.

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN

EVI<FNWYVDGVEVHNAI<TI<PREEQYASTYRVVSVLTVLHQDWLNGI<EYI<CT<V

SNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGGGSSGPVPLSLYSGSGGGAVNGTSQFTCFYNSRANISCVWSQD GALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVT LRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRACISWEISQASHY

FERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGE

FTTWSPWSQPLAFRTKPAALGKD (SEQ ID NO: 91)

Methods of Treatment

[0389] Provided herein are methods for treating or preventing a disease in a subject comprising administering to the subject an effective amount of any masked or targeted cytokine described herein or compositions thereof. In some embodiments, methods are provided for treating or preventing a disease in a subject comprising administering to the subject any composition described herein. In some embodiments, the subject (e.g., a human patient) has been diagnosed with cancer or is at risk of developing such a disorder. In some embodiments, methods are provided for treating or preventing disease in a subject comprising administering to the subject an effective amount of any masked or targeted cytokine described herein or compositions thereof, wherein the masked or targeted cytokine is activated upon cleavage by an enzyme. In some embodiments, the masked or targeted cytokine is activated at a tumor microenvironment. The masked or targeted cytokine is therapeutically active after it has cleaved. Thus, in some embodiments, the active agent is the cleavage product.

[0390] For the prevention or treatment of disease, the appropriate dosage of an active agent will depend on the type of disease to be treated, as defined herein, the severity and course of the disease, whether the agent is administered for preventive or therapeutic purposes, previous therapy, the subject’s clinical history and response to the agent, and the discretion of the attending physician. The agent is suitably administered to the subject at one time or over a series of treatments.

[0391] In some embodiments of the methods described herein, an interval between administrations of a masked or targeted cytokine described herein is about one week or longer. In some embodiments of the methods described herein, an interval between administrations of a masked or targeted cytokine described herein is about two days or longer, about three days or longer, about four days or longer, about five days or longer, or about six days or longer. In some embodiments of the methods described herein, an interval between administrations of a masked or targeted cytokine described herein is about one week or longer, about two weeks or longer, about three weeks or longer, or about four weeks or longer. In some embodiments of the methods described herein, an interval between administrations of a masked or targeted cytokine described herein is about one month or longer, about two months or longer, or about three months or longer. As used herein, an interval between administrations refers to the time period between one administration of the masked or targeted cytokine and the next administration of the masked or targeted cytokine. As used herein, an interval of about one month includes four weeks. In some embodiments, the treatment includes multiple administrations of the masked or targeted cytokine, wherein the interval between administrations may vary. For example, in some embodiments, the interval between the first administration and the second administration is about one week, and the intervals between the subsequent administrations are about two weeks. In some embodiments, the interval between the first administration and the second administration is about two days, three days, four days, or five days, or six days, and the intervals between the subsequent administrations are about one week.

[0392] In some embodiments, the masked or targeted cytokine is administered on multiple occasions over a period of time. The dosage that is administered to the subject on multiple occasions can, in some embodiments, be the same dosage for each administration, or, in some embodiments, the masked or targeted cytokine can be administered to the subject at two or more different dosages. For example, in some embodiments, a masked or targeted cytokine is initially administered at one dosage on one or more occasions and is later administered at a second dosage on one or more occasions beginning at a later time point.

[0393] In some embodiments, a masked or targeted cytokine described herein is administered at a flat dose. In some embodiments, a masked or targeted polypeptide described herein is administered to a subject at a dosage from about 25 mg to about 500 mg per dose. In some embodiments, the masked or targeted polypeptide is administered to a subject at a dosage of about 25mg to about 50mg, about 50mg to about 75mg, about 75mg to about lOOmg, about lOOmg to about 125mg, about 125mg to about 150mg, about 150mg to about 175mg, about 175mg to about 200mg, about 200mg to about 225mg, about 225mg to about 250mg, about 250mg to about 275mg, about 275mg to about 300mg, about 300mg to about 325mg, about 325mg to about 350mg, about 350mg to about 375mg, about 375mg to about 400mg, about 400mg to about 425mg, about 425mt to about 450mg, about 450mg, to about 475mg, or about 475mg to about 500mg per dose.

[0394] In some embodiments, a masked or targeted cytokine described herein is administered to a subject at a dosage based on the subject’s weight or body surface area (BSA). Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (e.g. 0.1 mg/kg-lOmg/kg) of masked or targeted cytokine can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the masked or targeted cytokine would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. In some embodiments, a masked or targeted polypeptide described herein is administered to a subject at a dosage from about 0.1 mg/kg to about 10 mg/kg or about 1.0 mg/kg to about 10 mg/kg. In some embodiments, a masked or targeted cytokine described herein is administered to a subject at a dosage of about any of 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5 mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 5.5 mg/kg, 6.0 mg/kg, 6.5 mg/kg, 7.0 mg/kg, 7.5 mg/kg, 8.0 mg/kg, 8.5 mg/kg, 9.0 mg/kg, 9.5 mg/kg, or 10.0 mg/kg. In some embodiments, a masked or targeted cytokine described herein is administered to a subject at a dosage of about or at least about 0.1 mg/kg, about or at least about 0.5 mg/kg, about or at least about 1.0 mg/kg, about or at least about 1.5 mg/kg, about or at least about 2.0 mg/kg, about or at least about 2.5 mg/kg, about or at least about 3.0 mg/kg, about or at least about 3.5 mg/kg, about or at least about 4.0 mg/kg, about or at least about 4.5 mg/kg, about or at least about 5.0 mg/kg, about or at least about 5.5 mg/kg, about or at least about 6.0 mg/kg, about or at least about 6.5 mg/kg, about or at least about 7.0 mg/kg, about or at least about 7.5 mg/kg, about or at least about 8.0 mg/kg, about or at least about 8.5 mg/kg, about or at least about 9.0 mg/kg, about or at least about 9.5 mg/kg, about or at least about 10.0 mg/kg, about or at least about 15.0 mg/kg, about or at least about 20mg/kg, about or at least about 30mg/kg, about or at least about 40mg/kg, about or at least about 50mg/kg, about or at least about 60mg/kg, about or at least about 70mg/kg, about or at least about 80mg/kg, about or at least about 90mg/kg, or about or at least about lOOmg/kg. Any of the dosing frequencies described above may be used.

[0395] A method of treatment contemplated herein is the treatment of a disorder or disease such as cancer with any of the masked or targeted cytokines or compositions described herein. Disorders or diseases that are treatable with the formulations of this present invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma) or testicular cancer.

[0396] In some embodiments, provided herein is a method of treatment or prevention of a cancer by administration of any masked or targeted cytokines or compositions described herein. In some embodiments, provided herein is a method of treatment or prevention of a cancer by administration of any masked or targeted cytokine or composition described herein in combination with an anticancer agent. The anti-cancer agent can be any agent capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell survival. In some embodiments, the anti -cancer agent is selected from the group consisting of a PD-1 inhibitor, an EGFR inhibitor, a HER2 inhibitor , a VEGFR inhibitor, a CTLA-4 inhibitor, a BTLA inhibitor, a B7H4 inhibitor, a B7H3 inhibitor, a CSFIR inhibitor, an HVEM inhibitor, a CD27 inhibitor, a KIR inhibitor, an NKG2A inhibitor, an NKG2D agonist, a TWEAK inhibitor, an ALK inhibitor, a CD52 targeting antibody, a CCR4 targeting antibody, a PD-L1 inhibitor, a KIT inhibitor, a PDGFR inhibitor, a BAFF inhibitor, an HD AC inhibitor, a VEGF ligand inhibitor, a CD 19 targeting molecule, a FOFR1 targeting molecule, a DFF3 targeting molecule, a DKK1 targeting molecule, a MUC1 targeting molecule, a MUG 16 targeting molecule, a PSMA targeting molecule, an MSFN targeting molecule, an NY-ES0-1 targeting molecule, a B7H3 targeting molecule, a B7H4 targeting molecule, a BCMA targeting molecule, a CD29 targeting molecule, a CD151targeting molecule, a CD 123 targeting molecule, a CD33 targeting molecule, a CD37 targeting molecule, a CDH19 targeting molecule, a CEA targeting molecule, a Claudin 18.2 targeting molecule, a CFEC12A targeting molecule, an EGFRVIII targeting molecule, an EPCAM targeting molecule, an EPHA2 targeting molecule, an FCRH5 targeting molecule, an FLT3 targeting molecule, a GD2 targeting molecule, a glypican 3 targeting molecule, a gpA33 targeting molecule, a GPRC5D targeting molecule, an IL-23R targeting molecule, an IL-1RAP targeting molecule, a MCSP targeting molecule, a RON targeting molecule, a ROR1 targeting molecule, a STEAP2 targeting molecule, a TfR targeting molecule, a CD 166 targeting molecule, a TPBG targeting molecule, a TROP2 targeting molecule, a proteasome inhibitor, an ABE inhibitor, a CD30 inhibitor, a FLT3 inhibitor, a MET inhibitor, a RET inhibitor, an IL- 1(3 inhibitor, a MEK inhibitor, a ROS1 inhibitor, a BRAE inhibitor, a CD38 inhibitor, a RANKE inhibitor, a B4GALNT1 inhibitor, a SLAMF7 inhibitor, an IDH2 inhibitor, an mTOR inhibitor, a CD20 targeting antibody, a BTK inhibitor, a PI3K inhibitor, a FLT3 inhibitor, a PARP inhibitor, a CDK4 inhibitor, a CDK6 inhibitor, an EGFR inhibitor, a RAF inhibitor, a JAK1 inhibitor, a JAK2 inhibitor, a JAK3 inhibitor, an IL-6 inhibitor, a IL- 17 inhibitor, a Smoothened inhibitor, an IL-6R inhibitor, a BCL2 inhibitor, a PTCH inhibitor, a PIGF inhibitor, a TGFB inhibitor, a CD28 agonist, a CD3 agonist, CD40 agonist, a GITR agonist, a 0X40 agonist, a VISTA agonist, a CD137 agonist, a LAG3 inhibitor, a TIM3 inhibitor, a TIGIT inhibitor, and an IL-2R inhibitor.

[0397] In some embodiments, provided herein is a method of treatment or prevention of a cancer by administration of any masked or targeted cytokine described herein in combination with an anti-inflammatory agent. The anti-inflammatory agent can be any agent capable of preventing, counteracting, inhibiting, or otherwise reducing inflammation.

[0398] In some embodiments, the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor. The COX inhibitor can be any agent that inhibits the activity of COX-1 and/or COX-2. In some embodiments, the COX inhibitor selectively inhibits COX-1 (i.e., the COX inhibitor inhibits the activity of COX-1 more than it inhibits the activity of COX- 2). In some embodiments, the COX inhibitor selectively inhibits COX-2 (i.e., the COX inhibitor inhibits the activity of COX-2 more than it inhibits the activity of COX-1). In some embodiments, the COX inhibitor inhibits both COX-1 and COX-2.

[0399] In some embodiments, the COX inhibitor is a selective COX-1 inhibitor and is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, and ketoprofen. In some embodiments, the COX inhibitor is a selective COX-2 inhibitor and is selected from the group consisting of celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, a chromene derivative, a chroman derivative, N-(2-cyclohexyloxynitrophenyl) methane sulfonamide, parecoxib, lumiracoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT- 2016, CT-3, ABT-963, SC-58125, nimesulide, flosulide, NS-398, L- 745337, RWJ-63556, L- 784512, darbufelone, CS-502, LAS-34475, LAS- 34555, S-33516, diclofenac, mefenamic acid, and SD-8381. In some embodiments, the COX inhibitor is selected from the group consisting of ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate. In some embodiments, the COX inhibitor is selected from the group consisting of SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, a chromene derivative, a chroman derivative, N-(2- cyclohexyloxynitrophenyl) methane sulfonamide, parecoxib, lumiracoxib, RS 57067, T- 614, BMS-347070, JTE-522, S-2474, SVT- 2016, CT-3, ABT-963, SC-58125, nimesulide, flosulide, NS-398, L- 745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS-34475, LAS- 34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate.

[0400] In some embodiments, the anti-inflammatory agent is an NF-KB inhibitor. The NF-KB inhibitor can be any agent that inhibits the activity of the NF-KB pathway. In some embodiments, the NF-KB inhibitor is selected from the group consisting of an IKK complex inhibitor, an IKB degradation inhibitor, an NF-KB nuclear translocation inhibitor, a p65 acetylation inhibitor, an NF-KB DNA binding inhibitor, an NF-KB transactivation inhibitor, and a p53 induction inhibitor.

[0401] In some embodiments, the IKK complex inhibitor is selected from the group consisting of TPCA-1, NF-KB Activation Inhibitor VI (BOT-64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, and IKK-16. In some embodiments, the IKB degradation inhibitor is selected from the group consisting of BAY-11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, and MLN-4924 (pevonedistat). In some embodiments, the NF-KB nuclear translocation inhibitor is selected from the group consisting of JSH-23 and rolipram. In some embodiments, the p65 acetylation inhibitor is selected from the group consisting of gallic acid and anacardic acid. In some embodiments, the NF-KB DNA binding inhibitor is selected from the group consisting of GYY-4137, p-XSC, CV-3988, and prostaglandin E2 (PGE2). In some embodiments, the NF-KB transactivation inhibitor is selected from the group consisting of LY-294002, wortmannin, and mesalamine. In some embodiments, the p53 induction inhibitor is selected from the group consisting of quinacrine and flavopiridol. In some embodiments, the NF-KB inhibitor is selected from the group consisting of TPCA-1, NF- KB Activation Inhibitor VI (BOT- 64), BMS-345541, amlexanox, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG-115, MG- 132, lactacystin, epoxomicin, parthenolide, carfilzomib, MLN-4924 (pevonedistat), JSH-23 rolipram, gallic acid, anacardic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wortmannin, mesalamine, quinacrine, and flavopiridol.

[0402] In some embodiments, provided herein is a method of treatment or prevention of a cancer by administration of any masked or targeted cytokine or composition described herein in combination with an anti-cancer therapeutic protein. The anti-cancer therapeutic protein can be any therapeutic protein capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell survival. Exemplary anti-cancer therapeutic proteins may come in the form of an antibody or fragment thereof, an antibody derivative, a bispecific antibody, a chimeric antigen receptor (CAR) T cell, a fusion protein, or a bispecific T-cell engager (BiTE). In some embodiments, provided herein is a method of treatment or prevention of a cancer by administration of any masked or targeted cytokine or composition described herein in combination with CAR-NK (Natural Killer) cells.

EXAMPLES

[0403] While certain compositions and methods of the present invention have been described with specificity in certain embodiments, the following examples serve only to illustrate the compositions and methods of the invention and are not intended to limit the same. Example 1: Engineered Masking Moiety

[0404] A masking moiety forms a part of a masked cytokine or targeted cytokine. The masking moiety masks the cytokine or its functional fragment in the masked cytokine or in the targeted cytokine, and thereby reducing or preventing binding of that cytokine or its functional fragment to its cognate receptor. The masking moiety disclosed herein includes an engineered CD 122 or a fragment thereof that masks the IL-2 cytokine.

[0405] Exemplary schematics of masked cytokines comprising a wild-type or an engineered CD122 masking moiety (the engineered CD122 is indicated with a star *) are shown in FIG. 1A. An exemplary schematics of masked IL-2 cytokine comprising an engineered CD122 masking moiety (indicated with a star (*)) with a cleavable linker is shown in FIG. IB.

[0406] The engineered CD 122 provided herein has been engineered for improved stability including thermal stability. To engineer CD 122 with improved stability, the following strategies have been employed: (a) searching for spatially proximal amino acid pairs that can facilitate disulfide bond formation, and mutating that amino acid to Cys; (b) not mutating free Cys to Ser; (c) looking at the locality and identifying native interactions and then choosing target amino acid; (d) searching for polar/charged amino acids in the hydrophobic core of domain DI and domain D2, and mutating them to “appropriate” hydrophobic residues if possible; and (e) searching for potential stabilizing charge-charge mutations to create salt bridges.

[0407] Using these methods, the present inventors have engineered the following mutations in the CD122: (a) C122S and C168S, (b) C122V and C168V, (c) C122A and C168V, (d) C168V, (e) C122V and C168A, (f) C122A and N123C, (g) C122V and N123C, (h) Cl 22 A and Cl 68 A, (i) VI 17C, N123Q and C168A, (j) N123Q, C168A, and L169C, (k) L106C, C122A, C168A and S195C (1) L106C, C122A, C168A and V184C (m) C122A, C168A, V184C, and S195C, (n) C122A, C168A, Q177C, and R204C, (o) L106C, C122V, C168V, and S195C or (p) F8C, A94C, C122V, and C168V. FIG. 1C illustrates the CD122 mutations generated in this example.

[0408] These engineered CD 122s were incorporated into a masked cytokine (MC) as represented in FIG. IB. Specifically, the masked cytokine comprises two polypeptide chains. The first chain comprises a first Fc polypeptide chain linked to an engineered CD122 via a cleavable linker. The first polypeptide chain comprises “hole mutations” (Y349C; T366S; L368A; and Y407V). The second chain comprises a second Fc polypeptide chain linked to an IL-2 cytokine. The second Fc polypeptide chain comprises “knob mutations” (S354C and T366W), and “RF mutations” (H435R and Y436F) in the CH3 domain. One or both of the Fc polypeptide can further comprise N297A. One of ordinary skill in the art can envision substituting the IL-2 cytokine with an IL- 15 cytokine, or any cytokine or protein that binds to CD 122.

Example 2: Engineered Masked Cytokines Demonstrating Improved Stability

[0409] In this example, masked cytokines comprising various engineered CD122 were tested for their stability. The masked cytokines tested in this example are shown in Table 1

Table 1. Masked Cytokine constructs and CD122 mutations

[0410] Fig. 2A illustrates the melting temperature (Tm) of masked cytokines comprising the engineered CD122, as measured by Differential Scanning Fluorimetry (DSF) in PBS at pH 7.4. DSF measures protein unfolding by monitory changes in fluorescence as a function of temperature. The results confirm that the engineered masked IL-2 cytokines displayed an improved stability and there was an increase of up to 4 °C in their melting temperature (Tm (°C)).

[0411] FIG. 2B illustrates DSF and Static Light Scattering (SLS) traces for MCI and MC6. The top traces illustrate the fluorescence ratio for the engineered masked IL-2 cytokines MCI and MC6 as a function of temperature. The bottom traces illustrate the intensity of scattered light for the engineered masked IL-2 cytokines MCI and MC6 as a function of temperature.

[0412] For DSF measurements, the MCI and MC6 were normalized to 1 mg/mL in 20 mM histidine, 150 mM NaCl at pH 6.0. The samples were heated from 25.0C to 95 °C at a heating rate of 1.0 °C/min. The melting temperature for MCI was 54.2 °C while the melting temperature for the MC6 was 57.1 °C.

[0413] For accelerated stability measurements, MCI and MC6 were normalized to 1 mg/mL in 20 mM histidine, 150 mM NaCl at pH 6.0, and stored them at 37 °C for incubation. At each time point, an aliquot was removed from 37 °C and stored at -80 °C. At the end of incubation, samples were subjected to size exclusion chromatography using a Zenix-C SEC-300 column (Sigma) using a flow rate of 0.35 mL/min.

[0414] FIG. 2C illustrates the accelerated stability of four of the masked cytokines comprising the engineered CD122 measured at day 0, day 3, day 7, and day 14 by a stress tolerance assay. The results show that the engineered CD122 displays significantly improved stability. Particularly, MC3, MC6, and MC14 exhibited below 10% high molecular weight species (HMWS) after 7 days, and below 20% even after 14 days.

Example 3: Masked Cytokines comprising Engineered Fc Domain

[0415] The masked cytokines of the present invention can be engineered to have improved stability and efficacy. In some embodiments, the Fc domain is engineered. In some embodiments, the cytokine is engineered. [0416] As illustrated in FIG. 1A and FIG. IB, a masked cytokine comprises a first Fc polypeptide and a second Fc polypeptide. The Fc region of the masked cytokines was further engineered in accordance with the strategies and methods described below.

Fc Domain Engineering

[0417] Fc domain was engineered for, among other applications, efficient purification of heterodimeric masked IL-2 cytokine. CH2 and CH3 domains of the first chain (“knob chain”) and the CH2 domain of the second chain (“hole chain”) were derived from either IgGl or IgG4. CH3 of the second chain was derived from either wild-type IgG3, or IgGl with mutations. The exemplary schematics are shown in FIG. 3. To assess if the heterodimeric Fc polypeptide can be purified without impurities, IL-2 cytokine and an IL-2 receptor p (IL-2RP; also known as CD122) was each fused to Fc chain as illustrated in FIG. 4A. The constructs shown in FIG. 4A were generated, purified, and characterized.

[0418] Plasmids encoding the constructs (e.g., native Fc, CH3 mutant, and chimeric Fc) were transfected into cells at different ratios of knob and hole chain (K:H of 1 : 1, 2: 1, and 1 :4 ). The Fc polypeptide chain was purified using protein A chromatography or by size exclusion chromatography (SEC). The samples after each protein A chromatography (“PostProA”) and SEC (“PostSEC”) were analysed by both non-reducing (NR) and reducing (R) gel electrophoresis. The impurities of different constructs were also assessed by measuring EC50 of active IL-2. As shown in FIG. 4A, the gel electrophoresis results show that residual “free knob” protein are present (~40KDa band) in both post Protein A and SEC purifications when cells were transfected with the native Fc format. However, both the CH3 mutant and the chimeric Fc formats successfully eliminated knob impurities. These findings were confirmed in a cell-based reporter assay (HEK Blue IL-2 assay) which determines % calculated active cytokine (FIG. 4B). As for the native Fc format, EC 50 value shifts depending on the amount of impurity (free Knob). On the other hand, EC50 remains unchanged for both CH3 mutant and the chimeric Fc formats due to lack of active impurity.

I l l Example 4: Engineered Targeted Masked Cytokine

[0419] The engineered CD 122 of the present invention can be incorporated in to a targeted cytokines. A targeted cytokine comprises a targeting moiety in addition to the masked cytokine described in Examples 2-3. Specifically, a targeted cytokine comprises a first Fc polypeptide, a second Fc polypeptide, a targeting moiety, a masking moiety, and a cytokine. Representative targeted cytokines (TC) are illustrated in FIG. 5 and FIG. 6A.

[0420] The targeting moiety of the engineered masked IL-2 cytokine was further engineered in accordance with the strategies and methods described below.

[0421] To examine whether the engineered Fc domains work in the context of the targeted cytokine, PD-1 targeting moiety fused to each knob and hole chain of the masked cytokine, generating a targeted cytokine (TC). In this example, four different targeted cytokine molecules (TC) were constructed and purity was determined by SEC, Nonreducing capillary electrophoresis sodium dodecyl sulfate (NR CE-SDS) and reducing capillary electrophoresis sodium dodecyl sulfate (Red CE-SDS), as shown in FIG. 5. The results show that the CH3 “RF” knob mutations effectively eliminated free knob in all targeted cytokine molecules tested.

Engineered Targeting Moiety

[0422] A targeting moiety can be engineered to be in different formats. For example, for bivalent targeting, a targeted IL-2 cytokine comprises 1) chain 1 with variable heavy and IgGl or IgG4 heavy constant region with hole mutations; 2) chain 2 with variable heavy and IgGl or IgG4 heavy constant with knob mutations and either includes CH3 of IgGl with “RF mutations” or IgG3 Ch3 domain, with IL-2 cytokine (or its variants) fused to the C-terminal; and 3) chain 3 with variable light and IgG kappa or lambda constant (FIG. 6A; Format A). For monovalent targeting, format Bl or B2 can be constructed, which comprises a target-specific Fab fused to either hole chain (Bl) or to knob chain (B2) through CHI domain. In this format, “hole chain” comprises Y349C/T366S/L368A/Y407V mutations, and “knob chain” has S354C/T366W mutations and either includes CH3 of IgGl with “RF mutations” or IgG3 Ch3 domain, with IL-2 cytokine (or its variants) fused to the C-terminal. Fab light chain is comprised of variable light and immunoglobulin kappa or lambda constant regions. Format C as shown in FIG. 6A can also be used for monovalent targeting, which comprises 1) chain 1 with a variable light and IgG kappa or lambda constant region fused to IgGl or 4 constant heavy region from upper hinge with “hole mutations” (Y349C/T366S/L368A/Y407V); 2) chain 2 with a variable light and IgG kappa or lambda constant region fused to variable heavy and IgGl or IgG4 heavy constant with knob mutations (S354C/T366W) and either includes CH3 of IgGl with “RF mutations” (H435R and Y436F) IgG3 Ch3 domain, with IL-2 cytokine (or its variants) fused to the C-terminal.

[0423] EC50 value of each construct was measured using the cell-based reporter assay (HEK Blue IL-2 assay), and the results show that IL-2 of all formats tested were active (FIG. 6B)

Example 5. On-Cell binding Assay of Targeted Cytokines with Engineered Masking moiety

[0424] In this example, binding of the targeted masked cytokines on CD3/CD28 activated CD8+ T cells was assessed.

[0425] The targeted masked cytokines shown in Table 5 were constructed. TC46 and TC47 comprises a modified CD122 that comprises stabilizing mutations.

Table 5. Features of Targeted Cytokines used in Example 7

[0426] Mixed donor lots of human peripheral blood mononuclear cells (PBMCs) were thawed and seeded in medium (RPMI1640, 10% fetal bovine serum (FBS), 2 mM 1- Glutamine, 50 U/mL penicillin, 50 pg/mL streptomycin, 1 pM each of non-essential amino acids, 10 mM HEPES, 1 mM sodium pyruvate, 55 pM B-mercaptoenthanol, 10 pg/mL gentamycin) into tissue culture treated plates that were coated for 1 h at 37°C with 1 pg/ml anti-CD3. Anti-CD28 was added in solution at a concentration of 5 pg/ml. Two days later the PBMCs were collected and transferred into a 96 well v-bottom plate at 100,000 cells per well in FACS Buffer (2% FBS in phosphate buffered saline (PBS)). Test articles were diluted to twice the indicated concentration in FACS buffer. Test articles were added to cells at a 1: 1 ratio to the final concentration indicated in the figure and incubated for 1 h at 4°C. The cells were washed three times with FACS buffer and fixed in 4%PFA. Cells were then washed once in FACS buffer. All cells were stained in 100 pL of indicated molecules in FACS buffer for 1 h at 4°C. The cells were washed three times with FACS buffer and fixed in 4% paraformaldehyde (PF A). All cells were subsequently stained in 100 pL with anti -human IgG PE, anti-CD3 PE/Dazzle 594, anti-CD45 PerCP Cy5.5, anti-CD8 BV510, and anti-CD4 BV421 for 30 minutes. Cells were washed twice to remove unbound antibody and analyzed on the BD Celesta by gating on CD3+CD8+ T cells. Data were fit and EC50s generated using a nonlinear regression [Agonist] vs. response - Variable slope (four parameter) in GraphPad Prism except for TKO and the isotype control which did produce full sigmoidal curves.

[0427] As shown in FIG. 7, all constructs bind CD3/CD28 activated CD8+ T cells with equivalent EC50 comparable to Pembrolizumab. Isotype controls exhibited limited to no binding at all concentrations tested.

Example 6: In Vivo Efficacy of Engineered Masked Cytokines

[0428] In vivo efficacies of various engineered masked cytokines is assessed by measuring anti-tumor activity in mice. The engineered masked cytokines in this example include constructs - MC1-16 (illustrated in FIG. 1C).

[0429] MC38 tumor cells is injected subcutaneously into the right flank of each mouse. Upon reaching -100 mm3 sized tumors (day 0), the mice receives a single intravenous dose of about 0.5-10 mg/kg of the construct. Samples are collected from various tissues (including tumor, spleen, blood) at various time points after dosing.

[0430] The proliferation of immune cells following treatment with engineered masked cytokine constructs is also assessed. Samples taken from tumor, spleen, and peripheral blood is assessed for the proliferation of CD8+ T cells. The tumor size and body weight are measured, among other things, to assess the efficacy of the masked cytokines of the present invention.

Example 7: In Vivo Efficacy of Engineered Targeted Masked Cytokines

[0431] In vivo efficacy of various Targeted Masked Cytokines is assessed by measuring anti-tumor activity in mice. The targeted masked cytokines that is tested in this example include targeted masked cytokine constructs. In some embodiments, a targeting moiety specifically binds PD-1. In some embodiments, a targeting moiety specifically binds PD-L1.

[0432] MC38 tumor cells is injected subcutaneously into the right flank of each mouse. Upon reaching -100 mm3 sized tumors (day 0), the mice receives a single intravenous dose of about 0.5-10 mg/kg of the construct. Samples are collected from various tissues (including tumor, spleen, blood) at various time points after dosing.

[0433] The proliferation of immune cells following treatment with engineered masked cytokine constructs is also assessed. Samples taken from tumor, spleen, and peripheral blood is assessed for the proliferation of CD8+ T cells. The tumor size and body weight are measured, among other things, to assess the efficacy of the targeted cytokines of the present invention.

EQUIVALENTS

[0434] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS An engineered CD122 polypeptide or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence. An engineered CD122 polypeptide or fragment thereof comprising a mutation at amino acid 122 and 168 relative to SEQ ID NO: 15, wherein the engineered CD122 has improved stability compared to a wild-type CD122. The engineered CD122 polypeptide of claim 1, comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 15. The engineered CD122 polypeptide of any one of claims 1-3, comprising a mutation to facilitate disulfide bond formation. The engineered CD122 polypeptide of any one of claims 1-3, comprising a mutation that creates a salt bridge. The engineered polypeptide of any one of claims 1-3, wherein the engineered CD122 comprises mutations selected from a) C122S and C168S, b) Cl 22V and Cl 68V, c) Cl 22 A and Cl 68V, d) Cl 68V, e) Cl 22V and Cl 68 A, f) C122A and N123C, g) C122V and N123C, h) C122A and C168A, i) VI 17C, N123Q and C168A, j) N123Q, C168A, and L169C, k) L106C, C122A, C168A and S195C l) L106C, C122A, C168A and V184C m) C122A, C168A, V184C, and S195C, n) C122A, C168A, Q177C, and R204C, o) L106C, C122V, C168V, and S195C or p) F8C, A94C, C122V, and Cl 68V. The engineered CD122 or fragment thereof of any one of claims 2-6, wherein the improved stability is measured by percent high molecular weight species (HMWS) or melting temperature. The polypeptide of any one of claims 1-7 wherein the engineered CD122 is fused to an Fc domain. A masked cytokine comprising: a) a cytokine or fragment thereof; b) a masking moiety comprising CD122 or a fragment thereof comprising one or more mutations selected from the group consisting of F8C, A94C, L106C, C122V, C122A, N123C, N123Q, C168V, C168A, L169C, Q177C, V184C, S195C, R204C relative to a wild-type CD122 amino acid sequence, and c) an Fc domain comprising a first Fc polypeptide linked to the masking moiety through a first linker and a second Fc polypeptide linked to the cytokine or a fragment thereof through a second linker, wherein the masking moiety binds to the cytokine or fragment thereof; and wherein the first or the second linker is a cleavable linker such that the masking moiety releases the cytokine or a fragment thereof upon cleavage. The masked cytokine of claim 9, wherein the masked cytokine has improved stability relative to a masked cytokine comprising a wild-type CD122. The masked cytokine of claim 9 or 10, wherein the CD122 comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 15. The masked cytokine of any one of claims 9-11, wherein the CD122 comprises a mutation to facilitate disulfide bond formation. The masked cytokine of any one of claims 9-12, wherein the CD122 comprises a mutation that creates a salt bridge. The masked cytokine of any one of claims 9-11, wherein the CD122 comprises mutations selected from a) C122S and C168S, b) Cl 22V and Cl 68V, c) Cl 22 A and Cl 68V, d) Cl 68V, e) Cl 22V and Cl 68 A, f) C122A and N123C, g) C122V and N123C, h) C122A and C168A, i) VI 17C, N123Q and C168A, j) N123Q, C168A, and L169C, k) L106C, C122A, C168A and S195C l) L106C, C122A, C168A and V184C m) C122A, C168A, V184C, and S195C, n) C122A, C168A, Q177C, and R204C, o) L106C, C122V, C168V, and S195C, or p) F8C, A94C, C122V, and Cl 68V. The masked cytokine of any one of claims 9-14, wherein the improved stability is measured by percent high molecular weight species (HMWS) or melting temperature. The masked cytokine of any one of claims 9-15, wherein the first or the second linker is a cleavable linker such that the masking moiety releases the IL-2 cytokine or a fragment thereof upon cleavage. The masked cytokine of any one of claims 9-16, wherein the linker comprises that is not cleaved comprises GGSSPPGGGSSGGGSG. The masked cytokine of any one of claims 9-17, wherein the cleavable linker comprises MPYDLYHP and/or VPLSLYSG. The masked cytokine of any one of 9-18, wherein the cleavable linker comprises between 8-50 amino acid residues. The masked cytokine of claim 9-18, wherein the cleavable linker has an in vitro cleavage efficiency yielding at least 10% active IL-2 cytokine. The masked cytokine of any one of the preceding claims, wherein the cleavable linker comprises a proteolytically cleavable peptide flanked on both sides by a spacer domain. The masked cytokine of claim 21, wherein the spacer domain is between 3 and 10 amino acids in length. The masked cytokine of claim 21, wherein the spacer domain is between 3 and 6 amino acids in length. The masked cytokine of any one of the preceding claims, wherein the first linker is the cleavable linker. The masked cytokine of any one of claims the preceding claims, wherein the second linker is the cleavable linker. The masked cytokine of any one of claims the preceding claims, wherein both the first linker and the second linker comprise a cleavable linker. The masked cytokine of any one of the preceding claims, wherein the C- terminus of the second Fc polypeptide is linked to the N-terminus of the masking moiety or a fragment thereof. The masked cytokine of any one of the preceding claims, wherein the C- terminus of the first Fc polypeptide is linked to the N-terminus of the IL-2 cytokine or a fragment thereof. The masked cytokine of any one of the preceding claims, wherein the Fc domain comprises s i. a Fc polypeptide comprising a CH3 domain comprising a modification that reduces or eliminates binding to Protein A, and ii. a Fc polypeptide comprising a CH3 domain that binds to Protein A. The masked cytokine of any one of the preceding claims, wherein Fc polypeptide comprising a CH3 domain that binds to Protein A comprises an IgGl, IgG2, or IgG4 Fc domain or a fragment thereof. The masked cytokine of any one of the preceding claims, wherein the Fc polypeptide comprising a CH3 domain comprising a modification that reduces or eliminates binding to Protein A comprises a first IgGl, IgG2, IgG3 or IgG4 Fc domain or a fragment thereof. The masked cytokine of claim 29-31, wherein the CH3 domain comprising a modification that reduces or eliminates binding to Protein A is a human IgGl, IgG2 or IgG4 sequence comprising a modification at position H435 and/or Y436 according to Kabat numbering. The masked cytokine of claim 32, wherein the modification is H435R and Y436F according to Kabat numbering. The masked cytokine of claim any one of the preceding claims, wherein the first and/ or the second Fc domains each contain one or more modifications that promote the non-covalent association of the first and the second Fc polypeptide. The masked cytokine of any one of the preceding claims, wherein a) the first Fc domain comprises Y349C; T366S, L38A, Y407V, and N297A mutations and the second Fc domain comprises S354C, T366W and N297A mutations; or b) the second Fc domain comprises Y349C; T366S, L38A, Y407V, and N297A mutations and the first Fc domain comprises S354C, T366W and N297A mutations. The masked cytokine of any one of the preceding claims, wherein a) the first Fc domain comprises Y349C, T366S, L38A, Y407V, N297A and I253A mutations and the second Fc domain comprises S354C, T366W, N297A and 1253 A mutations; or b) the second Fc domain comprises Y349C, T366S, L38A, Y407V, N297A and I253A mutations and the first Fc domain comprises S354C, T366W, N297A and 1253 A mutations. The masked cytokine of any one of the preceding claims, wherein the first or second Fc domain comprises SEQ ID NO: 86 and SEQ ID NO: 80. The masked cytokine of any one of claims 29-31, wherein the CH3 domain that reduces or eliminates binding to Protein A comprises a human IgG3 sequence. The masked cytokine of claim 38, wherein the first or second Fc domain comprises SEQ ID NO: 84 and SEQ ID NO: 80. The masked cytokine of any one of claims 9-39, wherein the cytokine or fragment thereof comprises an IL-2 or an IL-15 polypeptide or fragment thereof. The masked cytokine of claim 40, wherein the IL-2 cytokine or functional fragment thereof is modified compared to the sequence of a mature IL-2 having SEQ ID NO: 13. The masked cytokine of claim 41, wherein the modified IL-2 cytokine or functional fragment thereof comprises modifications R38A, F42A, Y45A, and E62A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. The masked cytokine of 41 or 42, wherein the modified IL-2 cytokine or functional fragment thereof comprises the modification C125A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. The masked cytokine of any one of claims 41-43, wherein the modified IL-2 cytokine or functional fragment thereof comprises R38A, F42A, Y45A, E62A and Cl 25 A relative to the sequence of a mature IL-2 having SEQ ID NO: 13. The masked cytokine of any one of claims 40-44, wherein the IL-2 cytokine or functional fragment thereof comprises an amino acid sequence of SEQ ID NO: 14. The masked cytokine of any one of claims 9-40, wherein cytokine comprises an IL- 15 cytokine or functional fragment thereof The masked cytokine of claim 46, wherein the IL- 15 polypeptide comprises an amino acid sequence of SEQ ID NO: 93 or an amino acid sequence having at least one amino acid modification as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, or E53 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions at positions D22, E46, E53, N71, N79, or N112 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N79 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises an amino acid sequence having an amino acid substitution at position N71, N79 and N112 as compared to the amino acid sequence of SEQ ID NO: 93. The masked cytokine of claim 47, wherein the IL- 15 cytokine or functional fragment thereof comprises any amino acid sequence of SEQ ID NO: 94-108. The masked cytokine of any one of the preceding claims further comprising a targeting moiety. The masked cytokine of claim 55, wherein the targeting moiety comprises one or more antigen binding domains, peptide, a polypeptide, a protein, a ligand, or an agent that specifically binds to the cytokine or the fragment thereof. The masked cytokine of claim 56, wherein the targeting moiety comprises an antigen binding domain selected from the group consisting of Fab, a single chain Fv (scFv), a single domain antibody (VHH), one or more CDRs, a variable heavy chain (VH), a variable light chain (VL), a Fab-like bispecific antibodies (bsFab), a single-domain antibody-linked Fab (s-Fab), an antibody, and a combination thereof. The masked cytokine of claim 57, wherein the targeted cytokine comprises a first antigen binding domain and a second antigen binding domain. The masked cytokine of claim 58, wherein the first and second antigen binding domains specifically bind to the same target. The masked cytokine of claim 59, wherein the first and second antigen binding domains comprise the same amino acid sequence. The masked cytokine of claim 58, wherein the first and second antigen binding domains specifically bind to different targets. The masked cytokine of claim 59 or 61, wherein the first and second antigen binding domains comprise different amino acid sequences. The masked cytokine of any one of claims 55-62, wherein the C-terminus of the first antigen binding domain is linked to the N-terminus of the first Fc polypeptide; wherein the C-terminus of the first Fc polypeptide is linked to the N- terminus of the cytokine or a fragment thereof; wherein the C-terminus of the second antigen binding domain is linked to the N-terminus of the second Fc polypeptide; and wherein the C-terminus of the second Fc polypeptide is linked to the N- terminus of the masking moiety. The masked cytokine of any one of claims 55-63, wherein the targeting moiety specifically binds PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, NKG2D, NKP44, NKP30, CD19, CD20, CD30, CD38, BCMA, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), delta-like protein 3 (DLL3), delta-like protein 4 (DLL4), epidermal growth factor receptor (EGFR), glypican-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGF Rl), vascular endothelial growth factor receptor 2 (VEG FR2), Nectin-4, Liv-1, glycoprotein NMB (GPNMB), prostate specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), six transmembrane epithelial antigen of the prostate 1 (STEAP1), folate receptor alpha (FR-a), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), ectonucleotide pyrophosphatase/phosphodiesterase family member 3 (ENPP3), mesothelin, trophoblast glycoprotein (TPBG), CD 19, CD20, CD22, CD33, CD40, CD56, CD66e, CD70, CD74, CD79b, CD98, CD 123, CD 138, CD352, CD47, signal -regulatory protein alpha (SIRPa), Claudin 18.2, Claudin 6, 5T4, fibroblast activation protein alpha (FAPa), the melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cellular adhesion molecule (EPCAM), or combinations thereof. The masked cytokine of claim 64, wherein the targeting moiety specifically binds PD-1. A nucleic acid encoding the engineered CD122 or masked cytokine of any one of the preceding claims. A vector comprising the nucleic acid of claim 66. A host cell comprising the nucleic acid of claim 67. A method of producing an engineered CD122 or masked cytokine comprising culturing the host cell of claim 68 under a condition that produces the engineered CD 122 or masked cytokine. A composition comprising the engineered CD122 or masked cytokine of any one of claims 1-65. A pharmaceutical composition comprising the engineered CD122 or masked cytokine of any one of claims 1-65, and a pharmaceutically acceptable carrier. A kit comprising the engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71. A method of treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71. A method of treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the engineered CD 122 or masked cytokine of any one of claims 1- 65, or the composition of claim 70, or the pharmaceutical composition of claim 71. An engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71, for use in medicine. An engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71, for use in treating a neoplastic disease. An engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71, for treating or preventing an inflammatory disease. An engineered CD122 or masked cytokine of any one of claims 1-65, or the composition of claim 70, or the pharmaceutical composition of claim 71, for use in treating or preventing an autoimmune disease.