Masked il-2 cytokines and methods of use thereof

Abstract

The present disclosure provides, among other things, a masked cytokine comprising an interleukin 2 (IL-2) polypeptide, a VHH masking moiety, an anti-PD1 targeting moiety, and an engineered Fc domain comprising a tumor-associated protease cleavage site. In such masked cytokines, the IL-2 polypeptide is engineered to be activatable at a target site, such as in a tumor microenvironment, by a protease. The VHH masking moiety blocks, occludes, inhibits (e.g., reduces), or otherwise prevents (e.g., masks) the activity or binding of the cytokine to its cognate receptor or protein. Upon proteolytic cleavage of the cleavage site in the Fc domain, the IL-2 polypeptide becomes activated, enabling it to bind to its cognate receptor or protein with increased affinity.

Description

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 619,623, filed January 10, 2024, and U.S. Provisional Application No. 63 / 689,277, filed August 30, 2024; the contents of each of the aforementioned U.S. Provisional Applications are incorporated herein by reference in their entirety.

[0002] Merging of sequence lists This application contains a sequence list submitted electronically in XML format. The entire contents of an XML file named “XTX_UNIVFC_03WO1_SL.xml”, created on December 23, 2024 and measuring 70,189 bytes, are hereby incorporated by reference. Background Technology

[0003] Cancer is the second leading cause of death in the United States, surpassing the next five leading causes of death (chronic respiratory diseases, stroke, accidents, Alzheimer's disease, and diabetes). While significant progress has been made in targeted therapies, much work remains to be done in this field. Immunotherapy and its branch, immuno-oncology, are creating viable and exciting treatment options for malignant tumors. Specifically, it is now recognized that a hallmark of cancer is immune evasion, and substantial efforts have been made to identify targets and develop therapies targeting these targets to reactivate the immune system to recognize and treat cancer.

[0004] One effective strategy for stimulating the immune system to induce anti-tumor cytotoxicity is cytokine therapy. Unfortunately, cytokines administered to patients typically have very short half-lives, necessitating frequent dosing. For example, the product label for aldesleukin, sold under the brand name Proleukin, indicates a half-life of 85 minutes in patients receiving a 5-minute intravenous (IV) infusion. Furthermore, high-dose cytokine administration can cause adverse health consequences, such as vascular leakage, through systemic immune activation. To prolong half-life and reduce toxicity, carrier moieties such as Fc domains, albumin, and PEG are fused to cytokines via cleavable linkers, allowing the release of active cytokines once the fused molecule reaches the tumor microenvironment. Summary of the Invention

[0005] The masked cytokine construct described herein comprises an IL-2 cytokine, a VHH masking moiety, a targeting moiety, and an engineered Fc domain. Specifically, the masked cytokine is engineered to optimize the binding between the anti-IL-2 VHH masking moiety and the IL-2 cytokine, thereby promoting safe and effective targeted therapy for cancer treatment. The VHH masking moiety binds to the IL-2 cytokine and inhibits the bioactivity of the cytokine in unintended targets. Furthermore, the Fc domain in the masked cytokine of this invention is engineered to cleave the substrate. Upon cleavage at the desired target (e.g., tumor), the masking moiety is released from the cytokine, thereby activating the function of the IL-2 peptide. Specifically, the masked cytokine comprising IL-2 and VHH masking fraction of the present invention is characterized by: (1) effective masking efficiency, such that the function of IL-2 cytokine is inhibited in undesired targets; (2) efficient activation of IL-2 by protease to release the VHH masking fraction; (3) selective activation of IL-2 in tumors rather than in plasma; and (4) in vivo efficacy (e.g., high tumor growth inhibition).

[0006] In one aspect, the present invention particularly provides a masked cytokine comprising an IL-2 polypeptide, a masking portion comprising a heavy-chain-only antibody (VHH), an anti-PD1 targeting portion, and an engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises a tumor-associated protease cleavage site and is fused to the IL-2 polypeptide or the masking portion such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

[0007] In one aspect, the present invention particularly provides a masked cytokine comprising an IL-2 polypeptide, a masking portion, a targeting portion, and an engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide contains a tumor-associated protease cleavage site between EU numbers 438-447, wherein the first Fc polypeptide is fused to the IL-2 polypeptide or the masking portion such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

[0008] In one aspect, the present invention particularly provides a masked cytokine comprising a weakened IL-2 polypeptide, a masking portion, a targeting portion, and an engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises a tumor-associated protease cleavage site of the sequence PLGL (SEQ ID NO: 1); wherein the first Fc polypeptide is fused to the IL-2 polypeptide or the masking portion such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

[0009] In some embodiments, the first Fc polypeptide comprises a tumor-associated protease cleavage site of the sequence SLPLGL (SEQ ID NO: 2). In some embodiments, the first Fc polypeptide comprises a tumor-associated protease cleavage site of the sequence GGPLGL (SEQ ID NO: 3).

[0010] In some implementations, the masking portion contains heavy chain-only antibodies (VHH).

[0011] In some implementations, the targeting portion is an anti-PD1 targeting portion.

[0012] In some embodiments, the targeting portion is scFv. In some embodiments, the targeting portion is Fab. In some embodiments, the targeting portion is VHH. In some embodiments, the targeting portion is an antigen-binding domain. In some embodiments, the targeting portion is a single-domain antibody (sdAb). In some embodiments, the targeting portion is a bispecific antibody.

[0013] In some embodiments, the tumor-associated protease cleavage site is located between EU numbers 444-447. In some embodiments, the tumor-associated protease cleavage site is located between EU numbers 440-447. In some embodiments, the tumor-associated protease cleavage site is located between EU numbers 438-447. In some embodiments, the tumor-associated protease cleavage site is located between EU numbers 438-446. In some embodiments, the tumor-associated protease cleavage site is located between EU numbers 442-447. In some embodiments, the tumor-associated protease cleavage site is located in the G chain of the Fc polypeptide.

[0014] In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence PLGL (SEQ ID NO: 1). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence SLPLGL (SEQ ID NO: 2). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence GGPLGL (SEQ ID NO: 3). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence MPY (SEQ ID NO: 4). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence MPYDLYHP (SEQ ID NO: 5). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence APAG (SEQ ID NO: 6). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence APAGLIVYN (SEQ ID NO: 7). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence PAN (SEQ ID NO: 8). In some embodiments, the tumor-associated cleavage site comprises the amino acid sequence PANLVAPDP (SEQ ID NO: 9).

[0015] In some embodiments, the engineered Fc peptide comprises amino acid substitutions of EU designations S442G, L443G, S444P, P445L, and G447L. In some embodiments, the engineered Fc peptide comprises amino acid substitutions of EU designations S444P, P445L, and G447L. In some embodiments, the engineered Fc peptide comprises amino acid substitutions of EU designations S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P. In some embodiments, the engineered Fc peptide comprises amino acid substitutions of EU designations Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N. In some embodiments, the engineered Fc peptide comprises amino acid substitutions of EU numbers Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

[0016] In some embodiments, IL-2 includes modifications R38A, F42A, Y45A, and E62A relative to the sequence of mature IL-2 having SEQ ID NO: 10. In some embodiments, IL-2 includes the modification C125A relative to the sequence of mature IL-2 having SEQ ID NO: 10. In some embodiments, IL-2 includes modifications F42E and C125A relative to the sequence of mature IL-2 having SEQ ID NO: 10. In some embodiments, IL-2 includes the modification F42E relative to the sequence of mature IL-2 having SEQ ID NO: 10.

[0017] In some embodiments, VHH comprises CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSFYEDETDY (SEQ ID NO: 16). In some embodiments, VHH comprises CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSFYEDETDY (SEQ ID NO: 17).

[0018] In one aspect, the present invention particularly provides a masked cytokine comprising: a weakened interleukin-2 (IL-2) polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16); an anti-PD1 targeting portion; and an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises amino acid substitutions of S442G, L443G, S444P, P445L, and G447L to engineer tumor-associated protease cleavage sites and the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and wherein the first Fc polypeptide is linked to the VHH masking portion and the Fc polypeptide is linked to the weakened IL-2 polypeptide.

[0019] In one aspect, the present invention particularly provides a weakened interleukin-2 (IL-2) polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSFYEDETDY (SEQ ID NO: 17); an anti-PD1 targeting portion; and an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises amino acid substitutions of S444P, P445L, and G447L to engineer tumor-associated protease cleavage sites and the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and wherein the first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the weakened IL-2 polypeptide.

[0020] In one aspect, the present invention particularly provides a weakened interleukin-2 (IL-2) polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16); an anti-PD1 targeting portion; and an Fc domain comprising a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc domain comprises amino acid substitutions of S444P, P445L, and G447L to engineer tumor-associated protease cleavage sites and the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and wherein the first Fc domain is linked to the VHH masking portion and the second Fc domain is linked to the weakened IL-2 polypeptide.

[0021] In one aspect, the present invention particularly provides a weakened interleukin-2 (IL-2) polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; CDR1 comprising the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 comprising the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR2 comprising the sequence ASSWYEDETDY (SEQ ID NO: 14). 16) The VHH masking portion of CDR3; the anti-PD1 targeting portion; and the Fc domain comprising a first Fc peptide and a second Fc peptide, wherein the first Fc domain comprises amino acid substitutions of S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P with engineered tumor-associated protease cleavage sites and the second Fc peptide does not contain tumor-associated protease cleavage sites; and wherein the first Fc domain is linked to the VHH masking portion and the second Fc domain is linked to a weakened IL-2 peptide.

[0022] In some embodiments, VHH includes one or more amino acid extensions at the C-terminus. In some embodiments, VHH includes an alanine extension at the C-terminus. In some embodiments, VHH includes the amino acid sequence AAA (SEQ ID NO: 18) at the C-terminus.

[0023] In some embodiments, VHH contains the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 19).

[0024] In some embodiments, VHH contains the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSSAAA (SEQ ID NO: 21).

[0025] In some embodiments, VHH contains the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKGRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAYASSFYEDETDYWGQGTQVTVSS (SEQ ID NO: 20).

[0026] In some embodiments, the targeting portion includes the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 43), the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 44), the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 45), the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 46), the light chain CDR2 sequence of LAS (SEQ ID NO: 47), and the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 48).

[0027] In some embodiments, the targeting portion comprises a heavy chain region having at least about 80% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 82% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 85% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 86% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 88% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 90% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 92% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having at least about 94% of the same amino acid sequence as SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 95% identical to that of SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 96% identical to that of SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 97% identical to that of SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 98% identical to that of SEQ ID NO: 41. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence of SEQ ID NO: 41.

[0028] In some embodiments, the targeting portion comprises a heavy chain region having at least about 80% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 82% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 85% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 86% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 88% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 90% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 92% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having at least about 94% of the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 95% identical to that of SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 96% identical to that of SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 97% identical to that of SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence that is at least about 98% identical to that of SEQ ID NO: 42. In some embodiments, the targeting portion comprises a heavy chain region having an amino acid sequence of SEQ ID NO: 42.

[0029] In some implementations, the targeting portion includes a heavy chain variable region containing SEQ ID NO: 41 and a light chain variable region containing SEQ ID NO: 42.

[0030] In some implementations, the second Fc polypeptide does not contain tumor-associated protease cleavage sites.

[0031] In some embodiments, a first Fc polypeptide is linked to an IL-2 polypeptide, and a second Fc polypeptide is linked to a masking portion.

[0032] In some embodiments, the first Fc polypeptide comprises amino acid substitutions of N297A. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of H435R and Y436F. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of Y349C, T366S, L368A, and Y407V. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of S354C and T366W. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of N297A, S354C, and T366W. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of N297A, S354C, T366W, H435R, and Y436F. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of N297A, Y349C, T366S, L368A, and Y407V. In some embodiments, the first Fc polypeptide comprises amino acid substitutions of N297A, Y349C, T366S, L368A, Y407V, H435R, and Y436F.

[0033] In some embodiments, the second Fc polypeptide contains amino acid substitutions of N297A. In some embodiments, the second Fc polypeptide contains amino acid substitutions of H435R and Y436F. In some embodiments, the second Fc polypeptide contains amino acid substitutions of Y349C, T366S, L368A, and Y407V. In some embodiments, the second Fc polypeptide contains amino acid substitutions of S354C and T366W. In some embodiments, the second Fc polypeptide contains amino acid substitutions of N297A, S354C, and T366W. In some embodiments, the second Fc polypeptide contains amino acid substitutions of N297A, S354C, T366W, H435R, and Y436F. In some embodiments, the second Fc polypeptide contains amino acid substitutions of N297A, Y349C, T366S, L368A, and Y407V. In some embodiments, the second Fc polypeptide comprises amino acid substitutions of N297A, Y349C, T366S, L368A, Y407V, H435R, and Y436F.

[0034] In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 23, and the second Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 24, and the second Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 25, and the second Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 26, and the second Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 27, and the second Fc polypeptide comprises SEQ ID NO: 33.

[0035] In some embodiments, the second Fc polypeptide comprises SEQ ID NO: 23, and the first Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the second Fc polypeptide comprises SEQ ID NO: 24, and the first Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the second Fc polypeptide comprises SEQ ID NO: 25, and the first Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the second Fc polypeptide comprises SEQ ID NO: 26, and the first Fc polypeptide comprises SEQ ID NO: 33. In some embodiments, the second Fc polypeptide comprises SEQ ID NO: 27, and the first Fc polypeptide comprises SEQ ID NO: 33.

[0036] In one aspect, the present invention particularly provides a masked cytokine comprising: an interleukin-2 (IL-2) polypeptide comprising an amino acid sequence at least 90% identical to that of SEQ ID NO: 13; a VHH masking portion comprising an amino acid sequence at least 90% identical to that of SEQ ID NO: 19; an anti-PD1 targeting portion comprising a variable heavy chain region (VH) at least 90% identical to that of SEQ ID NO: 41 and a variable light chain region (VL) at least 90% identical to that of SEQ ID NO: 42; a first Fc polypeptide comprising an amino acid sequence at least 90% identical to that of SEQ ID NO: 27; and a second Fc polypeptide comprising SEQ ID NO: 33, wherein the first Fc domain is linked to the VHH masking portion and the second Fc domain is linked to the IL-2 polypeptide.

[0037] In one aspect, the present invention particularly provides an interleukin-2 (IL-2) polypeptide comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 13; a VHH masking portion comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 21; an anti-PD1 targeting portion comprising a variable heavy chain region (VH) identical to that of SEQ ID NO: 41 and a variable light chain region (VL) identical to that of SEQ ID NO: 42; a first Fc polypeptide comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 27 or SEQ ID NO: 23; and a second Fc polypeptide comprising SEQ ID NO: 33, wherein the first Fc domain is linked to the VHH masking portion and the second Fc domain is linked to the IL-2 polypeptide.

[0038] In one aspect, the present invention particularly provides an interleukin-2 (IL-2) polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 12; a VHH masking portion comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 19; an anti-PD1 targeting portion comprising a variable heavy chain region (VH) that is at least 90% identical to that of SEQ ID NO: 41 and a variable light chain region (VL) that is at least 90% identical to that of SEQ ID NO: 42; a first Fc polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26; and a second Fc polypeptide comprising SEQ ID NO: 33, wherein the first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the IL-2 polypeptide.

[0039] In one aspect, the present invention particularly provides an interleukin-2 (IL-2) polypeptide comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 11; a VHH masking portion comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 19; an anti-PD1 targeting portion comprising a variable heavy chain region (VH) identical to that of SEQ ID NO: 41 and a variable light chain region (VL) identical to that of SEQ ID NO: 42; a first Fc polypeptide comprising at least 90% of the amino acid sequence identical to that of SEQ ID NO: 23; and a second Fc polypeptide comprising SEQ ID NO: 33, wherein the first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the IL-2 polypeptide.

[0040] In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 11. In some embodiments, the IL-2 polypeptide contains the same amino acid sequence as SEQ ID NO: 11 100%.

[0041] In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 12. In some embodiments, the IL-2 polypeptide contains the same amino acid sequence as SEQ ID NO: 12 100%.

[0042] In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 peptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide contains the same amino acid sequence as SEQ ID NO: 13 100%.

[0043] In some embodiments, the VHH masking portion contains at least 90% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 92% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 93% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 94% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 95% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 96% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 97% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 98% of the amino acids identical to those in SEQ ID NO: 19. In some embodiments, the VHH masking portion contains at least 99% of the amino acids identical to those in SEQ ID NO: 19. In some implementations, the VHH masking portion contains the same amino acid as SEQ ID NO: 19 100%.

[0044] In some embodiments, the VHH masking portion contains at least 90% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 92% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 93% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 94% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 95% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 96% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 97% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 98% of the amino acids identical to those in SEQ ID NO: 20. In some embodiments, the VHH masking portion contains at least 99% of the amino acids identical to those in SEQ ID NO: 20. In some implementations, the VHH masking portion contains the same amino acid as SEQ ID NO: 20 100%.

[0045] In some embodiments, the VHH masking portion contains at least 90% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 92% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 93% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 94% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 95% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 96% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 97% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 98% of the amino acids identical to those in SEQ ID NO: 21. In some embodiments, the VHH masking portion contains at least 99% of the amino acids identical to those in SEQ ID NO: 21. In some implementations, the VHH masking portion contains the same amino acid as SEQ ID NO: 21 100%.

[0046] In some embodiments, the VHH masking portion contains at least 90% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 92% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 93% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 94% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 95% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 96% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 97% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 98% of the same amino acids as SEQ ID NO: 22. In some embodiments, the VHH masking portion contains at least 99% of the same amino acids as SEQ ID NO: 22. In some implementations, the VHH masking portion contains the same amino acid as SEQ ID NO: 22 100%.

[0047] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 23. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 23 100%.

[0048] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 24. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 24 100%.

[0049] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 25. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 25 100%.

[0050] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 26. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 26 100%.

[0051] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 27. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 27 100%.

[0052] In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 90% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 92% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 93% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 94% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 95% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 96% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 97% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 98% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide comprises an amino acid sequence that is 99% identical to that of SEQ ID NO: 33. In some embodiments, the first Fc polypeptide contains the same amino acid sequence as SEQ ID NO: 33 100%.

[0053] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 63; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 74; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0054] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 64; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 66; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0055] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 66; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0056] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 63; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 66; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0057] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 68; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0058] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 69; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0059] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 70; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0060] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 67; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0061] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 65; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 71; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0062] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 63; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 72; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0063] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 63; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 73; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0064] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 63; a second polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 67; and a third polypeptide comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 50.

[0065] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 63. In some embodiments, the first polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 63.

[0066] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 64. In some embodiments, the first polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 64.

[0067] In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 65. In some embodiments, the first polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 65.

[0068] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 66. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 66.

[0069] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 67. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 67.

[0070] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 68. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 68.

[0071] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 69. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 69.

[0072] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 70. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 70.

[0073] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 71. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 71.

[0074] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 72. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 72.

[0075] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 73. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 73.

[0076] In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 74. In some embodiments, the second polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 74.

[0077] In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 50. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 50. In some embodiments, the second polypeptide comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 50. In some embodiments, the third polypeptide comprises an amino acid sequence that is 100% identical to that of SEQ ID NO: 50.

[0078] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 64; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0079] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0080] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0081] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 68; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0082] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 69; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0083] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 70; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0084] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 67; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0085] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 71; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0086] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 72; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0087] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 73; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0088] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 67; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0089] In one aspect, the present invention particularly provides a masked cytokine comprising: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 74; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0090] In one aspect, the present invention particularly provides a nucleic acid encoding the masking cytokines described herein.

[0091] In one aspect, the present invention provides a carrier comprising a nucleic acid encoding a masking cytokine described herein.

[0092] In one aspect, the present invention particularly provides a host cell comprising nucleic acids or a vector encoding the masking cytokines described herein.

[0093] In one aspect, the present invention particularly provides a method for producing the masked cytokines described herein, the method comprising culturing host cells under conditions for producing the masked cytokines.

[0094] In one aspect, the present invention particularly provides a pharmaceutical composition comprising the masking cytokines described herein and a pharmaceutically acceptable carrier.

[0095] In one aspect, the present invention particularly provides a kit comprising the masking cytokine and pharmaceutical composition described herein.

[0096] In one aspect, the present invention particularly provides a method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of the masking cytokine described herein.

[0097] In one aspect, the present invention particularly provides a method for treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of the masking cytokines described herein. Attached Figure Description

[0098] The accompanying drawings are for illustrative purposes only and are not intended to be limiting.

[0099] Figure 1 This is an exemplary schematic diagram of a cytokine-targeting construct containing a cleavable Fc domain. The crosshairs indicate the location of the cleavage site. One Fc domain is fused to a cytokine via an incleavable linker, and another Fc domain is fused to a masking motif (e.g., an anti-cytokine VHH) via an incleavable linker. Each Fc peptide also contains a targeting motif (e.g., Fab) that can specifically bind to a target (e.g., PD-1).

[0100] Figure 2A and Figure 2B An exemplary chart illustrating the percentage of STAT5 phosphorylation indicates the effectiveness of VHH masking in partially masking the IL-2 cytokine.

[0101] Figure 3A It showed that the tumor was caused by human tumors In vitro The percentage of masked IL-2 cytokines (UCM3, UCM9, and UCM11) cleaved. Figure 3B The percentage of masked IL-2 cytokines (UCM3, UCM9, and UCM11) cleaved from human plasma is shown. Figure 3C It showed that the tumor was caused by human tumors In vitro The percentage of masked IL-2 cytokines (UCM4, UCM5, UCM6, UCM2, UCM7, and UCM8) cleaved. Figure 3D The percentage of masked IL-2 cytokines (UCM4, UCM5, UCM6, UCM2, UCM7, and UCM8) cleaved from human plasma is shown. Figure 3E Showing fresh human tumor cells In vitro The percentage of masked IL-2 cytokine UCM11 cleaved. Figure 3F Showing fresh human tumor cells In vitro The percentage of masked IL-2 cytokine UCM12 cleaved.

[0102] Figure 4AA series of exemplary graphs illustrate the changes in tumor volume over time following treatment with the targeted IL-2 cytokines (UCM9, UCM11, and UCM3) of this invention. %TGI represents the tumor growth inhibition rate. Figure 4B A series of exemplary graphs illustrate overall survival rates following treatment with the IL-2-targeting cytokines (UCM9, UCM11, and UCM3) of the present invention. Figure 4C The illustrative graph shows the change in tumor volume over time after treatment with the targeted IL-2 cytokine (UCM2) of this invention. %TGI represents the tumor growth inhibition rate. Figure 4D The illustrative chart shows the overall survival rate after treatment with the IL-2 cytokine (UCM2) targeted by the present invention. Figure 4E The illustrative graph shows the change in tumor volume over time after treatment with the targeted IL-2 cytokine (UCM12) of this invention. %TGI represents the tumor growth inhibition rate. Figure 4F The illustrative chart shows the overall survival rate after treatment with the IL-2 cytokine (UCM12) of the present invention.

[0103] Figure 5A The illustrative graph shows the change in tumor volume over time after treatment with the targeted IL-2 cytokines (UCM4, UCM6, UCM7, and UCM8) of this invention. %TGI represents the tumor growth inhibition rate. Figure 5B The illustrative chart shows the overall survival rate after treatment with the IL-2-targeting cytokines (UCM4, UCM6, UCM7, and UCM8) of the present invention.

[0104] Figure 6A The exemplary SDS-PAGE image shows the cleavage of the masked cytokine UCM 12 by MMP9. Figure 6B A series of exemplary graphs show the percentage of STAT5 phosphorylation in UCM molecules with or without MMP9, and demonstrate the restoration of IL-2 activity by proteolytic cleavage of the VHH-masked portion. Figure 6C The cleavage of UCM12 by various proteases MMP1, MMP2, MMP7, MMP8 and MMP9 is shown. Figure 6D The image shows the CatL cutting of UCM12.

[0105] Figure 7 An exemplary graph showing reference subtracted light units (RSLU) indicates IL-2 activity as measured using HEK Blue IL-2 cells.

[0106] Figure 8The illustrative graph shows the fold induction of TCR signaling by an exemplary masked IL-2 cytokine in a PD-1 / PD-L1 blockade bioassay.

[0107] Figure 9 An exemplary pharmacokinetic analysis of the exemplary masked cytokine UCM12 in mice expressing human FcRn is shown.

[0108] Figure 10A Exemplary PBMC proliferation is shown with exemplary masking cytokines UCM3, UCM9, and UCM11. Figure 10B The study showed reduced proliferation of PBMCs of the exemplary masking cytokine UCM12 compared to the control UCM1.

[0109] definition To facilitate a clearer understanding of the invention, certain terms are defined below. Additional definitions for the following and other terms are set forth throughout the specification. Publications and other references cited herein to describe the background of the invention and to provide additional details on its practice are hereby incorporated by reference.

[0110] It should be understood that the present invention is not limited to any particular composition or biological system, which may of course be varied. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0111] As used in this specification and the appended claims, the singular forms “a / an” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “IL-2 polypeptide” optionally includes a combination of two or more such polypeptides, and so on.

[0112] As used herein, the term "about" refers to the typical range of error for a corresponding value that is readily known to those skilled in the art. References to "about" values ​​or parameters herein include (and describe) embodiments referring to said value or parameter itself.

[0113] It should be understood that the aspects and embodiments of the present invention described herein include “comprising aspects and embodiments,” “consisting of aspects and embodiments,” and “substantially consisting of aspects and embodiments.”

[0114] As used herein, the term “and / or” means any one of the items, any combination of items, or all of the items in connection with the term. For example, the phrase “A, B, and / or C” is intended to cover 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).

[0115] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies containing the Fc region of immunoglobulins), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, bispecific antibodies, and single-chain molecules), and antibody fragments (e.g., Fab, F(ab')2, and Fv). The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein.

[0116] The term "bibody antibody" refers to a small antibody fragment with two antigen-binding sites, which contains a heavy chain variable (VH) domain and a light chain variable (VL) domain linked together in the same polypeptide chain (VH-VL).

[0117] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called the J chain, containing 10 antigen-binding sites, while IgA antibodies contain 2–5 basic 4-chain units that can polymerize to combine with the J chain to form a multivalent assembly. In the case of IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is covalently disulfide-linked to the H chain, and two H chains are linked to each other by one or more disulfide bonds based on H chain isoforms. Each H and L chain also has regularly spaced intrachain disulfide bonds. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the a and y chains, and four CH domains for the p and s isoforms. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain at its other end. VL aligns with VH, and CL aligns with the first constant domain of the heavy chain (CHI). Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. VH and VL pair together to form a single antigen-binding site.

[0118] Based on the amino acid sequence of its constant domain, the L chain from any vertebrate species can be designated as one of two distinct types (called κ and λ). Immunoglobulins can be designated as different classes or isotypes based on the amino acid sequence of their heavy chain constant domain (CH). Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain designated as a, 8, e, y, and p, respectively. Classes y and a are further subdivided into subclasses based on relatively minor differences in CH sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants, referred to as allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol. 1, No. 4, pp. 1-7), any of which are applicable to this invention. Common allotypes in the human population are those denoted by the letters a, f, n, and z.

[0119] "Isolated" antibodies are antibodies identified, isolated, and / or recovered from components of their production environment (e.g., naturally or recombinantly). In some embodiments, the isolated peptide is unrelated to all other components in its production environment. Contaminant components in its production environment (such as contaminant components produced by recombinantly transfected cells) are substances that typically interfere with antibody research, diagnostic, or therapeutic uses and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the peptide: (1) is purified to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, greater than 99% by weight; (2) is purified to a degree sufficient to obtain at least 15 N-terminal or internal amino acid sequence residues using a rotary cup sequencer; or (3) is purified to homogeneity by SDS-PAGE staining under non-reducing or reducing conditions using Coomassie Brilliant Blue or silver staining. Isolated antibodies include in situ antibodies within recombinant cells because at least one component of the antibody's native environment is absent. However, isolated peptides or antibodies are typically prepared by at least one purification step.

[0120] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising said population are identical except for possible trace amounts of naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation). In some embodiments, the monoclonal antibody has 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 the 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, the monoclonal antibody has 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 the heavy chain and / or light chain. In some embodiments, the truncated form of the monoclonal antibody can be prepared by recombinant technology. In some embodiments, the monoclonal antibody is highly specific, targeting a single antigenic site. In some embodiments, the monoclonal antibody is highly specific, targeting multiple antigenic sites (such as bispecific or multispecific antibodies). The modifier "monoclonal" indicates the characteristics of an antibody obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibodies used according to the invention can be prepared by a variety of techniques, including, for example, hybridoma methods, recombinant DNA methods, phage display technology, and techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences.

[0121] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in essentially its complete form, rather than an antibody fragment. Specifically, whole antibodies include antibodies having both a heavy chain and a light chain containing an Fc region. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or a variant of its amino acid sequence. In some cases, intact antibodies may have one or more effector functions.

[0122] "Antibody fragment" includes a portion of a complete antibody, such as the antigen-binding region and / or variable region of a complete antibody, and / or the constant region of a complete antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of an Fc region, or a portion of an antibody containing an Fc region. Examples of antigen-binding antibody fragments include domain-bound antibodies (dAb), Fab, Fab', F(ab')2, and Fv fragments; bisomatic antibodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062

[1995] ); single-chain antibody molecules, and multispecific antibodies formed from antibody fragments, single-chain antibodies (scFv), single-domain antibodies (VHH), one or more CDRs, variable heavy chains (VH), variable light chains (VL), Fab-like bispecific antibodies (bsFab), Fab (s-Fab) linked with single-domain antibodies, and combinations thereof. Single-chain or single-light-chain antibodies can be engineered, or, in the case of heavy chains, isolated from camels, sharks, libraries, or mice engineered to produce single-chain molecules.

[0123] Antibody digestion with papain yields two identical antigen-binding fragments (referred to as "Fab" fragments) and a residual "Fc" fragment (the name reflecting its tendency to crystallize). The Fab fragment consists of the intact L chain, a variable region (VH) domain of the H chain, and a first constant domain (CHI) of the heavy chain. Each Fab fragment is monovalent for antigen binding, meaning it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to the two disulfide-linked Fab fragments with different antigen-binding activities and is still capable of cross-linking the antigen. The Fab' fragment differs from the Fab fragment in that it has additional residues at the carboxyl terminus of the CHI domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the nomenclature for Fab' used in this paper, where one or more cysteine ​​residues in the constant domain have a free thiol group. The F(ab')2 antibody fragments are initially produced as a pair of Fab' fragments with a hinge cysteine ​​residue between them. Other chemical conjugation methods for antibody fragments are also known. The Fc fragment comprises the carboxyl-terminal portion of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by the sequence and glycans in the Fc region, which is also recognized by Fc receptors (FcRs) on certain types of cells.

[0124] The "percentage of amino acid sequence identity (%)" for a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and the introduction of gaps (if necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. Alignment for determining the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, for example, 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 sequence alignment, including any algorithms required to achieve maximum alignment across the full length of the compared sequences. For example, the percentage of amino acid sequence identity for a given amino acid sequence A pair and / or for a given amino acid sequence B (or can be expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity for a given amino acid sequence B) is calculated as follows: 100 multiplied by the fraction X / Y Where X is the number of amino acid residues that are identified as identical matches when the program compares A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity percentage between A and B will not be equal to the amino acid sequence identity percentage between B and A.

[0125] Antibody "effector functions" refer to those biological activities attributable to the Fc region of the antibody (the native Fc region or the Fc region of an amino acid sequence variant), and these functions vary with antibody isotypes. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0126] "Attenuated IL-2" is used herein to refer to an IL-2 variant containing one or more mutations that reduce, but do not eliminate, binding to IL-2Rα. The inventors have found that "attenuated IL-2" expands tumor-specific T cells more effectively than "non-α IL-2" and exhibits lower regulatory T cell activity than its "α-biased IL-2" counterpart. In some embodiments, attenuated IL-2 contains mutations in F42E and C125A.

[0127] “Non-α IL-2” is used herein to refer to an IL-2 variant that includes one or more mutations that eliminate binding to IL-2Rα. In some embodiments, non-α IL-2 includes mutations of R38A, F42A, Y45A, E62A, and C125A relative to wild-type IL-2.

[0128] "α-biased IL-2" is used in this document to refer to an IL-2 variant that contains one or more mutations that reduce or eliminate binding to IL-2Rβγ. α-biased IL-2 has a "biased" affinity for cells that constitutively express IL-2α (e.g., regulatory T cells).

[0129] As used herein, “binding affinity” refers to the strength of the non-covalent interaction 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 for binding proteins (e.g., cytokines) is typically expressed as a dissociation constant (Kd). Affinity can be measured using methods commonly known in the art, including those described herein.

[0130] The “isolated” nucleic acid molecule encoding the cytokine peptides described herein is a nucleic acid molecule that has been identified and isolated from at least one contaminant nucleic acid molecule that is typically associated with it in the environment in which it is produced. In some embodiments, the isolated nucleic acid is unrelated to all components associated with the production environment. The isolated nucleic acid molecules encoding the peptides and cytokine peptides described herein are in a form or arrangement different from those found or set in nature. Therefore, the isolated nucleic acid molecules are distinguished from the nucleic acids encoding the peptides and cytokine peptides described herein, which are naturally present in cells.

[0131] The term "pharmaceutical formulation" refers to a formulation in which the biological activity of the active ingredient is permitted and which does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0132] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations used. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; 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 dextrin; 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™.

[0133] As used herein, the term "treatment" refers to a clinical intervention aimed at altering the natural processes of the treated individual or cells during the course of a clinical lesion. Desired therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and mitigating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a condition (e.g., a neoplastic disease) are improved or eliminated. Similarly, an individual is successfully "treated" if treatment improves the quality of life of a patient with a disease, reduces the dosage of other medications required to treat the disease, reduces the frequency of disease recurrence, alleviates the severity of the disease, delays the development or progression of the disease, and / or prolongs the individual's survival.

[0134] As used in this article, "in combination with" or "in combination with" refers to the application of one treatment method at the same time as the application of another treatment method. Therefore, "in combination with" or "in combination with" means the application of one treatment method before, during, or after the application of another treatment method to an individual.

[0135] As used herein, the term "prevention" includes providing prevention of the onset or recurrence of disease in an individual. An individual may be susceptible to, at risk of developing, or at risk of developing a certain condition, but has not yet been diagnosed with that condition. In some implementations, the targeted cytokines described herein are used to delay the progression of disease.

[0136] As used herein, an individual at “risk” for a disease may or may not have a detectable disease or symptoms of a disease, and may or may not have exhibited a detectable disease or symptoms of a disease prior to the treatments described herein. “At risk” means that an individual has one or more risk factors that are measurable parameters associated with the development of a disease as known in the art. Individuals with one or more of these risk factors have a higher probability of developing the disease than individuals without one or more of these risk factors.

[0137] "Effective dose" means a dose and time period that is at least effective in achieving the desired or indicated effect, including therapeutic or preventative outcomes.

[0138] Effective doses may be provided in one or more administrations. A “therapeutic effective dose” is the minimum concentration required to at least affect a measurable improvement in a specific condition. The therapeutic effective dose described herein may vary depending on factors such as the patient’s disease state, age, sex, and weight, as well as the antibody’s ability to elicit the desired response in an individual. A therapeutic effective dose may also be the amount by which any toxic or adverse effects of the targeted cytokine are offset by the beneficial effects of the treatment. A “prophylactic effective dose” refers to the effective dose and time period required to achieve the desired preventative outcome. Typically, but not always, the prophylactic effective dose may be less than the therapeutic effective dose because the prophylactic dose is administered to the subject before or in an early stage of the disease.

[0139] "Chronic" administration refers to the continuous rather than acute application of one or more drugs in order to prolong the period of time that the initial therapeutic effect (activity) is maintained. "Intermittent" administration is treatment that is not continuous and uninterrupted, but is inherently periodic.

[0140] As used herein, "individual" or "subject" refers to a mammal. "Mammalian" used for therapeutic purposes includes humans, livestock and farm animals, as well as zoo, sport, or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, etc. In some implementations, the individual or subject is a human.

[0141] Amino acids: As used herein, the 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, amino acids have the universal structure H₂N–C(H)(R)–COOH. In some embodiments, amino acids are naturally occurring amino acids. In some embodiments, amino acids are synthetic amino acids; in some embodiments, amino acids are d-amino acids; and in some embodiments, amino acids are l-amino acids. "Standard amino acid" refers to any of the twenty standard l-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than standard amino acids, whether synthetic or derived 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 substitutes. Amino acids (including carboxyl and / or amino-terminal amino acids in peptides) can be modified by methylation, amidation, acetylation, protecting groups, and / or substitution with other chemical groups that can alter the cyclic half-life of the peptide without adversely affecting its activity. Amino acids can participate in the formation of disulfide bonds. Amino acids may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprene-like 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 can refer to free amino acids and / or amino acid residues of peptides. It will be apparent from the context in which the term is used that it refers to either free amino acids or peptide residues.

[0142] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). 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, a genetically engineered animal, and / or a cloned animal.

[0143] Bioactivity: As used herein, the phrase “bioactivity” refers to the property of any agent that is active in a biological system, particularly in an organism. For example, an agent that has a biological effect on an organism when administered to it is considered to be bioactive.

[0144] Delivery: As used herein, the term “delivery” encompasses both local delivery and systemic delivery. For example, mRNA delivery encompasses the situation where mRNA is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also known as “local distribution” or “local delivery”), as well as the situation where mRNA is delivered to a target tissue and the encoded protein is expressed and secreted into the patient’s circulatory system (e.g., serum) and distributed systemically and absorbed by other tissues (also known as “systemic distribution” or “systemic delivery”).

[0145] Dosing interval: As used herein, in the context of a method for treating a disease, a dosing interval is the frequency at which a therapeutic composition (e.g., an mRNA composition) is administered in a subject (mammal) of need at an effective dose of mRNA, such that one or more symptoms associated with the disease are reduced; or one or more biomarkers associated with the disease are reduced for a period of time at least the dosing interval. Dosing frequency and dosing interval are used interchangeably in this disclosure.

[0146] Expression: As used herein, “expression” of a nucleic acid sequence means the translation of mRNA into a polypeptide, the assembly of multiple polypeptides into a complete protein (e.g., an enzyme), and / or post-translational modifications of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production”, as well as grammatical equivalents, are used interchangeably.

[0147] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to values ​​relative to baseline measurements, such as measurements taken on the same individual prior to initiation of the treatment described herein, or measurements taken on control subjects (or multiple control subjects) in the absence of the treatment described herein. A “control subject” is a subject who suffers from the same form of disease as the treated subject and whose age is approximately the same as the treated subject.

[0148] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc., rather than in a multicellular organism.

[0149] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0150] Patient: As used herein, the terms "patient" or "subject" refer to any organism to which the provided composition may be administered, for example, for experimental, diagnostic, preventative, 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, the patient is a human. Humans include both prenatal and postnatal forms.

[0151] Pharmaceutically acceptable: As used herein, “pharmaceutically acceptable” means a substance that, within a reasonable range of medical diagnoses, is suitable for contact with tissues in humans and animals without excessive toxicity, irritation, allergic reactions or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0152] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, non-toxic acid addition salts are salts of amino groups 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 salts of amino groups formed by using other methods used in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogen sulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate-heptate, glycerophosphate, gluconate, hemisulfate, heptaate, hydroiodate, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectate, persulfate, 3-phenylpropionate, phosphate, picrate, pentanoate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, etc. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts, and N+(C1-4 alkyl)4 salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Where appropriate, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions (such as halide, hydroxide, carboxyl, sulfate, phosphate, nitrate, sulfonate, and arylsulfonate ions). Further pharmaceutically acceptable salts include those formed by quaternizing amines with suitable electrophilic agents such as alkyl halides to form quaternized alkylamine salts.

[0153] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, meaning a person who seeks medical attention from a healthcare provider for the diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may have a disease or condition or be susceptible to a disease or condition, but may or may not exhibit symptoms of said disease or condition.

[0154] Essentially: As used herein, the term “essentially” refers to a qualitative condition that exhibits a target characteristic or property of general or near-general extent or degree. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if any) reach completion, and / or proceed to completion or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to describe the inherent potential lack of completeness in many biological and chemical phenomena.

[0155] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by the disease to be treated. In some implementations, target tissue includes those tissues exhibiting disease-related pathology, symptoms, or features.

[0156] Treatment: As used herein, the term "treatment" means any method used to partially or completely relieve, improve, alleviate, suppress, prevent, delay the onset, reduce the severity, and / or decrease the incidence of one or more symptoms or features of a particular disease, condition, and / or disorder. Treatment may be administered to subjects who do not exhibit signs of disease and / or only exhibit early signs of disease in order to reduce the risk of developing disease-related pathology.

[0157] Various aspects of the present invention will be described in detail in the following sections. The use of sections is not intended to limit the invention. Each section may be applied to any aspect of the invention. In this application, unless otherwise stated, the use of "or" means "and / or". Detailed Implementation

[0158] The masked cytokine constructs described herein are engineered to optimize the binding between the masking moiety (e.g., anti-IL-2 VHH) and the cytokine (e.g., IL-2 peptide) to facilitate a safe and effective targeted therapeutic agent for cancer treatment. The VHH masking moiety binds to the IL-2 cytokine and inhibits the cytokine's bioactivity in unintended targets. Furthermore, the Fc domain in the masked cytokine of this invention is engineered to contain a cleavage substrate. Upon cleavage in the desired target (e.g., tumor), the masking moiety is released from the cytokine, activating the function of the IL-2 peptide. Specifically, the masked cytokine of this invention comprising IL-2 and the VHH masking moiety is characterized by: (1) efficient masking efficiency, resulting in inhibition of IL-2 cytokine function in unintended targets; (2) efficient activation of IL-2 by a protease to release the VHH masking moiety; (3) selective activation of IL-2 in tumors, rather than in plasma; and (4) in vivo efficacy (e.g., high tumor growth inhibition).

[0159] Interleukin-2 (IL-2) This document provides an IL-2 polypeptide or a functional fragment thereof for use in any masked cytokine or its cleavage product. IL-2 plays a crucial role in cell signaling, particularly in cells of the immune system, by regulating leukocyte activity. The IL-2 polypeptide suitable for use in this invention can be any IL-2 polypeptide or a functional fragment thereof. In some embodiments, the IL-2 polypeptide is naturally occurring IL-2. In some embodiments, the IL-2 polypeptide comprises one or more substitutions (e.g., IL-2 mutants or IL-2 variants), or truncated IL-2. In some embodiments, IL-2 is a polypeptide that retains at least one property of IL-2 biological activity. In this application, the terms IL-2 polypeptide and IL-2 cytokine are used interchangeably.

[0160] In eukaryotic cells, the naturally occurring IL-2 polypeptide is synthesized as a 153-amino acid precursor polypeptide. It is then processed into mature IL-2 by removing amino acid residues 1-20. This yields the mature form of IL-2, which consists of 133 amino acids (amino acid residues 21-153) as shown in SEQ ID NO: 10. In some embodiments, the IL-2 polypeptide is naturally occurring IL-2.

[0161] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO:10) In some embodiments, the mature IL-2 peptide contains a mutation at position 125 relative to SEQ ID NO: 10. In some embodiments, the mutation contains C125A, resulting in SEQ ID NO: 11.

[0162] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:11) weakened IL-2 The inventors have discovered that using "attenuated IL-2" more effectively expands tumor-specific T cells than other IL-2 variants (e.g., "non-α IL-2") and exhibits lower regulatory T cell activity than other IL-2 variants (e.g., "α-biased IL-2"). "Attenuated IL-2" refers to an IL-2 variant containing one or more mutations compared to wild-type IL-2, said one or more mutations reducing but not eliminating binding to IL-2Rα. "Non-α IL-2" refers to an IL-2 variant containing one or more mutations eliminating binding to IL-2Rα, while "α-biased IL-2" refers to an IL-2 variant containing one or more mutations reducing or eliminating binding to IL-2Rβγ. In some embodiments, attenuated IL-2 contains the F42E mutation. In some embodiments, attenuated IL-2 contains both the F42E and C125A mutations.

[0163] In some embodiments, amino acid substitution reduces the affinity of the IL-2 peptide or a functional fragment thereof for CD25 (IL-2Rα). In some embodiments, the IL-2 peptide or a functional fragment thereof contains a mutation that reduces, but does not eliminate, the binding affinity for IL-2Ra (e.g., attenuated IL-2).

[0164] In some embodiments, the IL-2 peptide or its functional fragment comprises an amino acid sequence having one or more amino acid substitutions compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 10, thereby reducing the affinity of the IL-2 peptide or its functional fragment for IL-2Rα (CD25).

[0165] In some embodiments, the IL-2 peptide or a functional fragment thereof contains an amino acid sequence substitution F42E compared to the amino acid sequence of SEQ ID NO: 10. In some embodiments, the IL-2 peptide or a functional fragment thereof contains an amino acid sequence substitution F42E compared to the amino acid sequence of SEQ ID NO: 11.

[0166] In some embodiments, the IL-2 peptide or a functional fragment thereof contains amino acid sequence substitutions F42E and C125A compared to the amino acid sequence of SEQ ID NO: 10.

[0167] In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 13.

[0168] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTEKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:13) In some embodiments, the IL-2 polypeptide or a 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 with the amino acid sequence of SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 80% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 13. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 13.

[0169] In some embodiments, the IL-2 peptide includes a substitution at position 42 relative to SEQ ID NO: 10. In some embodiments, the substitution is F42A. In some embodiments, the substitution is F42E. In some embodiments, the substitution is F42K. In some embodiments, the substitution is F42S. In some embodiments, the substitution is F42R. In some embodiments, the substitution is F42Q. In some embodiments, the substitution is F42Y.

[0170] In some embodiments, the IL-2 peptide comprises a sequence selected from Table 1. In some embodiments, the IL-2 peptide comprises SEQ ID NO: 10. In some embodiments, the IL-2 peptide comprises SEQ ID NO: 11. In some embodiments, the IL-2 peptide comprises SEQ ID NO: 13.

[0171] The “functional fragment” of the IL-2 polypeptide comprises a portion of the full-length cytokine protein that retains or has modified cytokine receptor binding capacity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to the full-length cytokine protein). Cytokine receptor binding capacity can be characterized, for example, the ability of a cytokine to bind to a homologous receptor of the cytokine or a component thereof (e.g., one or more chains of a heterotrimeric receptor complex).

[0172] In some embodiments, the IL-2 peptide comprises “non-α IL-2”. “Non-α IL-2” is used herein to refer to an IL-2 variant comprising one or more mutations that eliminate binding to IL-2Rα. In some embodiments, the non-α IL-2 comprises mutations of R38A, F42A, Y45A, E62A, and C125A relative to wild-type IL-2. In some embodiments, the IL-2 peptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 12.

[0173] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:12) In some embodiments, the IL-2 peptide or a functional fragment thereof is any naturally occurring interleukin-2 (IL-2) protein or a modified variant thereof capable of binding to the interleukin-2 receptor (particularly the IL-2Rα chain). In the context of IL-2 peptide binding, the target protein may be IL-2R (including IL-2Rα, IL-2Rβ, and IL-2Rγ chains), the IL-2Rα chain, the IL-2Rβ chain, or the IL-2Rα / β dimer complex.

[0174] VHH masking section This invention particularly provides novel anti-IL-2 antigen-binding fragments (e.g., VHH) that can be used as a masking portion of any masked or targeted IL-2 cytokine described herein. The VHH masking portion binds to the IL-2 cytokine and inhibits the cytokine's biological activity. Upon cleavage, the masking portion is released from the cytokine, activating the function of the IL-2 peptide. Specifically, the VHH masking portion of this invention is characterized by improved binding kinetics, efficient IL-2 masking efficiency (i.e., inhibition of IL-2 in a non-target environment), and activation of IL-2 activity after release from IL-2 to promote anti-tumor responses in the tumor microenvironment. The VHH masking portion of this invention effectively masks or inhibits IL-2 activity, resulting in an EC50 value based on pSTAT5 activity on CD8+ T cells with masked IL-2 cytokines that is 300 times greater than that of IL-2 cytokines without the masking portion. In some embodiments, the masked cytokine has an EC50 value based on pSTAT5 activity on CD8+ T cells that is 300 times higher than that of the IL-2 cytokine without the masking portion. However, IL-2 function is restored upon cleavage of the masking portion from the IL-2 cytokine molecule. Preferably, in at least one cancer indication tested, the percentage of cleaved molecules in the tumor is greater than 5%, and the percentage of cleaved molecules in the plasma is below the lower limit of quantitation (LLOQ).

[0175] In some embodiments, the anti-IL-2 VHH of the present invention has improved stability, developability, and manufacturability for large-scale production for therapeutic use. In some embodiments, the anti-IL-2 VHH of the present invention has reduced immunogenicity (i.e., inducing minimal endotoxin or having reduced binding to pre-existing antidrug antibodies).

[0176] In some implementations, anti-IL-2 VHH binds to wild-type IL-2, attenuated IL-2, and non-α IL-2 with high affinity (e.g., ≤ 5 nM).

[0177] In some embodiments, the masking portion is a heavy chain-only antibody (VHH). In some embodiments, the masking portion is the anti-IL-2 VHH antibody described herein.

[0178] VHH antibodies (or nanobodies) are antigen-binding fragments of heavy-chain antibodies only. VHHs can be derived from organisms that produce VHH antibodies, such as camels and sharks. In some cases, VHHs can be recombinant VHHs. VHH technology is based on fully functional antibodies from camels that lack light chains. These heavy-chain antibodies contain a single variable domain (V... H VHH comprises a single-chain polypeptide having three CDRs and four frame regions (FR1-4). As used herein, a "frame region" or "FR" refers to a region located within a variable domain between CDRs. As used herein, a "complementarity-determining region" or "CDR" refers to a variable region in VHH containing an amino acid sequence capable of specifically binding to an antigen target (e.g., cytokines). In this application, VHH and sdAb are used interchangeably.

[0179] In some implementations, the VHH comprises three CDRs and four frame regions, named FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some implementations, the VHH may be truncated at the N-terminus or C-terminus, such that it contains only a portion of FR1 and / or FR4, or lack one or both of those frame regions, as long as the VHH substantially maintains cytokine binding and specificity.

[0180] In some embodiments, the VHH masking portion is connected to the Fc structural domain. In some embodiments, the VHH masking portion is connected to the cuttable Fc structural domain via a non-cuttable joint. In some embodiments, the VHH masking portion is connected to the non-cuttable Fc structural domain via a non-cuttable joint.

[0181] In some embodiments, the VHH antibody binds to the IL-2 peptide. In some embodiments, the VHH antibody binds to wild-type IL-2. In some embodiments, the VHH antibody binds to a variant of IL-2. In some embodiments, the VHH antibody binds to an engineered variant of IL-2. In some embodiments, the VHH antibody binds to attenuated IL-2. In some embodiments, the VHH antibody binds to non-α IL-2.

[0182] In some embodiments, the VHH masking portion of the present invention is further modified. Some modifications provide the modified molecule with a reduced ability to bind to a pre-existing antidrug antibody (ADA) compared to the unmodified molecule. In other words, the modified molecule will bind to the pre-existing ADA with reduced affinity or cohesion. Modifications can be selected from, for example, but not limited to, C-terminal addition, amplification, deletion, or tagging. Additional modifications are described in WO2013024059A2, the contents of which are incorporated herein by reference.

[0183] Furthermore, compared to unmodified molecules that do not contain C-terminal extensions, additions, deletions, or tags, modified molecules may have an enhanced safety profile and fewer side effects. Administration of pharmaceutical compositions containing modified molecules can produce improved immunogenicity and increased efficacy. Compositions containing modified molecules can be advantageously used for repeated dosing in subjects who may develop autoantibodies against unmodified molecules.

[0184] In some embodiments, the targeted masking cytokine includes modifications. In some embodiments, the targeted masking cytokine includes C-terminal extension. In some embodiments, the targeted masking cytokine includes C-terminal addition. In some embodiments, the targeted masking cytokine includes C-terminal deletion. In some embodiments, the targeted masking cytokine includes a C-terminal tag.

[0185] In some embodiments, the masking portion includes embellishments. In some embodiments, the masking portion includes a C-terminal extension. In some embodiments, the masking portion includes a C-terminal addition. In some embodiments, the masking portion includes a C-terminal deletion. In some embodiments, the masking portion includes a C-terminal label.

[0186] In some embodiments, the VHH antibody contains modifications. In some embodiments, the VHH antibody contains a C-terminal extension. In some embodiments, the VHH antibody contains a C-terminal addition. In some embodiments, the VHH antibody contains a C-terminal deletion. In some embodiments, the VHH antibody contains a C-terminal tag.

[0187] In some embodiments, the C-terminal extension comprises alanine. In some embodiments, the C-terminal extension comprises the sequence AS (SEQ ID NO: 34). In some embodiments, the C-terminal extension comprises the sequence AST (SEQ ID NO: 35). In some embodiments, the C-terminal extension comprises the sequence AAA (SEQ ID NO: 18). In some embodiments, the C-terminal extension comprises the sequence AH (SEQ ID NO: 36). In some embodiments, the C-terminal extension comprises the sequence GS (SEQ ID NO: 37). In some embodiments, the C-terminal extension comprises the sequence PP (SEQ ID NO: 38). In some embodiments, the C-terminal extension comprises the sequence PPP (SEQ ID NO: 39). In some embodiments, the C-terminal extension comprises the sequence GP (SEQ ID NO: 40). In some embodiments, the C-terminal modification comprises a deletion and an extension comprising the sequence GP (SEQ ID NO: 40).

[0188] In some embodiments, the VHH antibody comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises the amino acid sequence of SEQ ID NO: 22.

[0189] Table A. Exemplary VHH Masking Section (underlined in CDR) In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 87% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 19. In some embodiments, the VHH antibody comprises the same amino acid sequence as SEQ ID NO: 19.

[0190] In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 87% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 20. In some embodiments, the VHH antibody comprises the same amino acid sequence as SEQ ID NO: 20.

[0191] In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 87% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 93% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 21. In some embodiments, the VHH antibody comprises the same amino acid sequence as SEQ ID NO: 21.

[0192] In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 85% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 87% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 92% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 93% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 94% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 96% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 97% identical to SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 22. In some embodiments, the VHH antibody comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 22. In some embodiments, the VHH antibody comprises the same amino acid sequence as SEQ ID NO: 22.

[0193] Table B. CDR of an exemplary VHH masking portion In some implementations, the VHH antibody comprises CDR1 of GSIFSINVMG (SEQ ID NO: 14), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of ASSWYEDETDY (SEQ ID NO: 16).

[0194] In some implementations, the VHH antibody comprises CDR1 of GSIFSINVMG (SEQ ID NO: 14), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of ASSFYEDETDY (SEQ ID NO: 17).

[0195] In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by one amino acid. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by two amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by three amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by four amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by five amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by six amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by seven amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 14 by 8 amino acids.

[0196] In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by one amino acid. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by two amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by three amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by four amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by five amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by six amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by seven amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by 8 amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by 9 amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 15 by 10 amino acids.

[0197] In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by one amino acid. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by eight amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by three amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by four amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by five amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by six amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by seven amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by 8 amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by 9 amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 16 by 10 amino acids.

[0198] In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by one amino acid. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by eight amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by three amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by four amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by five amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by six amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by seven amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by 8 amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by 9 amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 17 by 10 amino acids.

[0199] Cuttable Fc domain carrier portion A longer in vivo half-life is important for therapeutic proteins. Unfortunately, cytokines administered to subjects typically have short half-lives because they are usually rapidly eliminated from the subject's body through mechanisms including renal clearance and endocytic degradation. Therefore, in the masked cytokines presented herein, the carrier portion is linked to the cytokine or the masking portion, with the aim of prolonging the cytokine's half-life in vivo, etc.

[0200] As used herein, the term "cleavable carrier" refers to any agent that can be enzymatically or non-enzymatically cleaved from the compositions of the present invention. In the context of this disclosure, the terms "cleavable carrier" and "cleavable domain" are used interchangeably. The cleavable carrier portion of the present invention provides enzymatically induced prodrug activation, characterized in that the cleavage of the carrier portion controls the release of the active therapeutic agent. The carrier portion may be an Fc domain, wherein at least one tumor-associated protease cleavage site is engineered, for example, by amino acid substitution at a specific position within the Fc domain.

[0201] In some implementations, the carrier portion is an Fc domain.

[0202] According to this disclosure, the carrier portion is an Fc domain derived from immunoglobulins such as IgM, IgG1, IgG2, IgG3, IgG4, IgD, IgE, and IgA, or variants thereof or fragments thereof. Therefore, the Fc domain is genetically engineered to make it enzymatically cleavable. The engineered Fc domain is referred to as a "cleavable Fc domain."

[0203] In some embodiments, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide. In some embodiments, the Fc domain is derived from any antibody or fragment thereof comprising a heavy-chain Fc polypeptide or a light-chain Fc polypeptide. In some embodiments, the Fc domain comprises a portion of a heavy-chain polypeptide or a light-chain polypeptide. In some embodiments, the antibody or a fragment thereof comprises an Fc domain or a fragment thereof. In some embodiments, the Fc domain derived from the antibody or a fragment thereof comprises a hinge region, a CH2 domain, and a CH3 domain, or fragments thereof. In some embodiments, the Fc domain comprises only the constant domain of the heavy-chain polypeptide. In some embodiments, the Fc domain comprises only the constant domain of the light-chain polypeptide. In some embodiments, the Fc domain comprises a first Fc polypeptide having CH2 and CH3 domains and a second Fc polypeptide having CH2 and CH3 domains. In some embodiments, the Fc domain comprises a first Fc polypeptide having a CH3 domain and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain comprises a first Fc polypeptide having CH2 and CH3 domains and a second Fc polypeptide having a CH3 domain. In some embodiments, the Fc domain comprises a first Fc polypeptide having a CH3 domain and a second Fc polypeptide having both CH2 and CH3 domains.

[0204] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are linked via a linker (such as a short peptide linker). In some embodiments, the linker is non-cleavable.

[0205] In some embodiments, the Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 28.

[0206] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 28).

[0207] In some embodiments, the Fc domain comprises an amino acid sequence that is at least 85% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 88% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 92% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 94% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 96% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 97% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO: 28. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 99% identical to that of SEQ ID NO: 28.

[0208] Introduction of cuttable substrates An engineered cuttable Fc domain contains one or more cuttable sites.

[0209] In some embodiments, the Fc domain comprises a first Fc polypeptide containing a cleavage site and a second Fc polypeptide not containing a cleavage site. In some embodiments, the Fc domain comprises a first Fc polypeptide not containing a cleavage site and a second Fc polypeptide containing a cleavage site. In some embodiments, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein each of the first and second Fc polypeptides contains a cleavage site.

[0210] As used herein, a “cleavage site” refers to a recognizable cleavage site on a portion of a cleavable peptide found in any Fc domain. A “cleavage site” can be an amino acid sequence, such as a short peptide motif, that is recognized and cleaved by a cleaving agent. A cleavage site may be a naturally occurring amino acid sequence within an Fc domain. Additionally and / or alternatively, cleavage sites may be introduced into the cleavable portion of an Fc domain by mutations (e.g., amino acid insertions, substitutions, and deletions). Mutations do not alter other activities of the Fc domain. In some embodiments, one or more cleavage peptide motifs may be engineered into the cleavable Fc domain as described herein. The cleaving agent may be an enzyme, such as a protease. In some embodiments, the cleavage site is a protease cleavage site, such that the cleavable Fc domain can be proteatically cleaved. A protease is an enzyme that cleaves and hydrolyzes the peptide bond between two specific amino acid residues of a target substrate protein. Proteases typically recognize specific peptide motifs and cleave the peptide bond between two specific amino acid residues within a short peptide motif.

[0211] The Fc domain can be engineered to contain one or more peptide motifs that can be recognized by one or more proteases, such as tumor-associated proteases, tissue-selective proteases, and disease-associated proteases (e.g., inflammation). As a non-limiting example, the cleavable Fc domain described herein can be cleaved by disease-associated or tissue-selective proteases selected from: matrix metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, and MMP28), cathepsins (cathepsin B, cathepsin D, and cathepsin...). The following enzymes are listed: cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S and cathepsin W), ADAM, ADAMTS, kallikrein 1 to 15, HTRA 1-2-3, HGFAc, PRSS, TMPRSS, elastase, PR-3, granzymes (granzymes A, B, M, H and K), fibroblast activating protein (FAP), plasmin, urokinase plasminogen activator (uPA), trypsin, caspase, thrombin, legumin, chymase, collagenase, aspartic protease A (napsin A), and matriptase 1-2.

[0212] In some embodiments, the cleavable Fc domain, as described herein, includes at least one engineered tumor-associated protease cleavage site. A "tumor-associated protease cleavage site" as provided herein is an amino acid sequence recognized by the protease, the expression of which is specific to or upregulated in tumor cells or their tumor cell environment. In some embodiments, the tumor-associated protease is a matrix metalloproteinase (MMP) selected from the group consisting of: MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP27, and MMP28. In one embodiment, the tumor-associated protease is MMP2. In another embodiment, the tumor-associated protease is MMP3. In yet another embodiment, the tumor-associated protease is MMP9. In yet another embodiment, the tumor-associated protease is MMP10. In some embodiments, the protease is cathepsin B. In other embodiments, the protease is an interstitial protease.

[0213] The advantage of this type of engineered cleavable Fc domain is that it provides a universal design architecture; the fusion of the engineered cleavable Fc domain with the therapeutic agent and subsequent cleavage at the cleavage site allow for the release of the agent in a specific environment (e.g., the tumor microenvironment).

[0214] The tumor cell environment is complex and may contain a variety of different proteases. Therefore, the precise location where the Fc domain is cleaved in the tumor cell environment can vary depending on the tumor type, between patients with the same tumor type, and even between the cleavage products formed within the same tumor, depending on the specific tumor cell environment. Furthermore, even after cleavage, the initial cleavage product may be further modified by the further action of proteases in the tumor cell environment, for example, by removing one or two terminal amino acids. It is anticipated that upon administration of a single-structure targeted cytokine as described herein, the distribution of the cleavage product will occur within the patient's tumor cell environment.

[0215] It should be understood that, as mentioned herein, a cleavage site refers to a site within a cleavable peptide between two specific amino acid residues that are known targets of proteases associated with the tumor cell environment. In this sense, there may be more than one cleavage site within a cleavable peptide as described herein, with different proteases cleaving the peptide at different cleavage sites. There may also be more than one protease acting on the same cleavage site within a cleavable peptide. Discussions of protease cleavage sites can be found in the art.

[0216] Therefore, the cleavable Fc domain disclosed herein can be cleaved by one or more proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more tumor-associated proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more tissue-selective proteases. In some embodiments, the cleavage site of the cleavable Fc domain is a cleavage site of one or more inflammation-associated proteases. In other embodiments, the cleavable Fc domain comprises one or more cleavage sites of other disease-associated proteases.

[0217] In some implementations, the Fc domain can be cleaved as a substrate for a protease, which is co-localized in regions, tissues, or organs expressing cytokine receptors.

[0218] In some embodiments, the length of the cleavable peptide motif in the Fc domain is 3 to 18 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 5 to 10 amino acids, or 5 to 8 amino acids, or 6 to 10 amino acids, or 7 to 10 amino acids, or 6 to 12 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 3 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 4 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 5 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 6 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 7 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 8 amino acids. In some embodiments, the length of the cleavable peptide motif in the Fc domain is 9 amino acids. In some implementations, the length of the cleavable peptide motif in the Fc domain is 10 amino acids.

[0219] In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is located in the hinge region, CH2 domain, CH3 domain, and / or CH2-CH3 domain linker region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is located in the F chain region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is located in the FG loop region. In some embodiments, the protease cleavage site within the engineered cleavable Fc domain is located in the G chain region.

[0220] In some implementations, the cleavable sites within the engineered cleavable Fc domain can be created based on short amino acid motifs located near the cleavage sites of the protease.

[0221] In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises PLGL (SEQ ID NO: 1). In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises SLPLGL (SEQ ID NO: 2). In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises GGPLGL (SEQ ID NO: 3). In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises MPYDLYHP (SEQ ID NO: 5). In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises APAGLIVPYN (SEQ ID NO: 7). In some embodiments, the engineered cleavable peptide motif within the cleavable Fc domain comprises PANLVAPDP (SEQ ID NO: 9).

[0222] In some embodiments, the engineered cleavable Fc domain includes one or more substitutions in the CH3 domain. In some embodiments, the engineered cleavable Fc domain includes one or more substitutions in the C-terminal region within the Fc domain. In some embodiments, the engineered cleavable Fc domain includes one or more substitutions in the G chain within the Fc domain. In some embodiments, the engineered cleavable Fc domain includes one or more substitutions at positions between EU numbers 436-447. In some embodiments, the engineered cleavable Fc domain includes one or more substitutions at positions between EU numbers 438 and 447.

[0223] In some embodiments, the cleavable peptide motif is located between EU numbers 438 and 445. In some embodiments, the cleavable peptide motif is located between EU numbers 439 and 446. In some embodiments, the cleavable peptide motif is located between EU numbers 440 and 447. In some embodiments, the cleavable peptide motif is located between EU numbers 438 and 447. In some embodiments, the cleavable peptide motif is located between EU numbers 437 and 444. In some embodiments, the cleavable peptide motif is located between EU numbers 440 and 443. In some embodiments, the cleavable peptide motif is located between EU numbers 441 and 444. In some embodiments, the cleavable peptide motif is located between EU numbers 442 and 445. In some embodiments, the cleavable peptide motif is located between EU numbers 444 and 447. In some embodiments, the cleavable peptide motif is located between EU numbers 441 and 447. In some embodiments, the cleavable peptide motif is located between EU numbers 443 and 447. In some embodiments, the cleavable peptide motif is located between EU numbers 436 and 443. In some embodiments, the cleavable peptide motif is located between EU numbers 442 and 447.

[0224] In some implementations, the engineered cuttable Fc domain includes one or more of the substitutions shown in Table C.

[0225] Table C. Exemplary engineered Fc domains containing cuttable substrates In some embodiments, the engineered cutable Fc domain is replaced by EU designations S444P, P445L, and G447L. In some embodiments, the engineered cutable Fc domain is replaced by EU designations S442G, L443G, S444P, P445L, and G447L. In some embodiments, the engineered cutable Fc domain is replaced by EU designations S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P. In some embodiments, the engineered cutable Fc domain is replaced by EU designations Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N. In some implementations, the engineered cuttable Fc domain includes replacements with EU designations Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

[0226] In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises EU-designated substitutions of S444P, P445L, and G447L. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises EU-designated substitutions of S442G, L443G, S444P, P445L, and G447L. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises EU-designated substitutions of S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises substitutions of EU numbers Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the first Fc polypeptide comprises substitutions of EU numbers Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

[0227] In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises substitutions of EU numbers S444P, P445L, and G447L. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises substitutions of EU numbers S442G, L443G, S444P, P445L, and G447L. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises substitutions of EU numbers S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises substitutions of EU numbers Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N. In some embodiments, the engineered cleavable Fc domain comprises a first Fc polypeptide and a second Fc polypeptide, wherein the second Fc polypeptide comprises substitutions of EU numbers Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

[0228] In some implementations, the engineered cleavable Fc domain is used as a carrier moiety for cytokines used in cancer therapy. The engineered cleavable Fc domain is fused to a masking cytokine molecule such that when an engineered protease cleavage site (such as an engineered tumor-associated protease cleavage site in the cleavable Fc domain) is cleaved, the masking cytokine is released from the masking moiety.

[0229] In some implementations, the cleavable Fc domain may include, in addition to incorporating one or more protease cleavage sites, other mutations described herein.

[0230] Fc engineered to promote heterodimerization or increase half-life Fc domains or fragments thereof capable of FcRn-mediated recycling can reduce or otherwise delay the clearance of targeted cytokines from a subject, thereby prolonging the half-life of the administered targeted cytokines. In some embodiments, the cleavable Fc domain or fragment thereof is any antibody or fragment thereof capable of FcRn-mediated recycling, such as any heavy chain polypeptide or portion thereof (e.g., an Fc domain or fragment thereof) capable of FcRn-mediated recycling.

[0231] The cleavable Fc domain or a fragment thereof can be derived from any antibody or fragment thereof. However, in some embodiments, the first or second Fc polypeptide may not bind to the FcRn receptor, such as a light chain polypeptide. For example, in some embodiments, the first Fc polypeptide does not directly interact with the FcRn receptor, but the targeted cytokine still has an extended half-life due to the inclusion of a second Fc polypeptide capable of interacting with the FcRn receptor (e.g., by including a heavy chain polypeptide). It is recognized in the art that FcRn-mediated recycling requires the binding of the FcRn receptor to the Fc region of an antibody or fragment thereof. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU indexing system) are important for the interaction between the human Fc region and the human FcRn complex. See, for example, Firan, M. et al., Int. Immunol. 13 (2001) 993-1002; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604. Various mutants of residues 248-259, 301-317, 376-382, and 424-437 (numbered according to the Kabat EU index numbering system) have also been examined and reported. Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0232] In some embodiments, the first and / or second Fc polypeptides capable of cleaving the Fc domain each contain one or more modifications to promote non-covalent association between the first and second Fc polypeptides. In some embodiments, the first Fc polypeptide comprises an IgG1 Fc domain or a fragment thereof, the IgG1 Fc domain or fragment thereof containing mutations Y349C; T366S; L368A; and Y407V to form a "mortar" in the first half-life extension domain, and the second Fc polypeptide comprises an IgG1 Fc domain or a fragment thereof, the IgG1 Fc domain or fragment thereof containing mutations S354C and T366W to form a "pestle" in the second half-life extension domain.

[0233] In some embodiments, the first and second Fc peptides are each an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof. In some embodiments, the first and second Fc peptides are each an IgG1 Fc domain or a fragment thereof.

[0234] In some embodiments, the first and second Fc peptides comprise SEQ ID NO: 28 having an amino substitution to promote association between the first Fc peptide and the second Fc peptide according to the "mortar and pestle" method.

[0235] In some embodiments, the sequence of SEQ ID NO: 28 contains mutations S354C and T366W (numbered according to the Kabat EU numbering system) to form a "pestle" in the first Fc polypeptide, and mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system) to form a "mortar" in the second Fc polypeptide.

[0236] In some embodiments, the sequence of SEQ ID NO: 28 contains mutations Y349C; T366S; L368A; and Y407V (numbered according to the Kabat EU numbering system) to form a “mortar” in the first Fc polypeptide, and mutations S354C and T366W (numbered according to the Kabat EU numbering system) to form a “pestle” in the second Fc polypeptide. These modified sequences have SEQ ID NOs: 29 and 30 as shown below: Fc peptides with mortises (Y349C; T366S; L368A; and Y407V): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 29) Fc polypeptides with club-like structures (S354C and T366W): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 30) In some embodiments, the first and second Fc polypeptides each further comprise an amino-substituted N297A, numbered according to the Kabat EU numbering system: Fc peptides with mortises (Y349C; T366S; L368A; Y407V and N297A): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 31) Fc polypeptides containing clubs (S354C, T366W, and N297A): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 32) 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 with the amino acid sequence of SEQ ID NO: 29.

[0237] 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 with the amino acid sequence of SEQ ID NO: 30.

[0238] 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 with the amino acid sequence of SEQ ID NO: 31.

[0239] 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 with the amino acid sequence of SEQ ID NO: 32.

[0240] 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 with the amino acid sequence of SEQ ID NO: 29.

[0241] 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 with the amino acid sequence of SEQ ID NO: 30.

[0242] 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 with the amino acid sequence of SEQ ID NO: 31.

[0243] 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 with the amino acid sequence of SEQ ID NO: 32.

[0244] In some embodiments, the first Fc polypeptide comprises an amino acid sequence having one or more modifications (such as substitution, addition, or deletion of one or more amino acids) compared to the amino acid sequence of any of SEQ ID NO: 29-32. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having one or more modifications (such as substitution, addition, or deletion of one or more amino acids) compared to the amino acid sequence of any of SEQ ID NO: 29-32. 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 the polypeptide chain and / or inhibit homodimerization of the polypeptide chain, alter effector function, or enhance effector function.

[0245] In some implementations, the protease cleavage site, as described herein, may be incorporated into any of SEQ ID NO: 29-32.

[0246] In some embodiments, the cleavable Fc domain may further comprise one or more amino acid substitutions, thereby altering effector function. In some embodiments, the half-life extension domain is the IgG1 Fc domain or a 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, P331S, S267E, L328F, D265A, and P329G, numbered according to the Kabat EU numbering system. In some embodiments, the half-life extension domain is an IgG2 Fc domain or a fragment thereof, and contains one or more amino acid substitutions: 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 a fragment thereof, and contains 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 a fragment thereof, and contains one or more amino acid substitutions: L235A, G237A, and E318A; S228P, L234A, and L235A; H268Q, V309L, A330S, and P331S; 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 a fragment thereof, and contains 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.

[0247] In some embodiments, the cleavable Fc domain further comprises one or more amino acid substitutions, thereby enhancing effector function. In some embodiments, the half-life extension domain is IgG1. Fc domain or fragments thereof, and containing one or more amino acid substitutions: S298A, E333A, and K334A; S239D and I332E; S239D, A330L, and I332E; P247I and A339D or A339Q; D280H and K290S; D280H, K290S, and 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 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, Q311M, and A 330M or A330F; K334V, S298T, and A330M or A330F; K334V, S239D, and A330M or S298T; L234Y, Y296W, and K290Y, F243V or E294L; Y296W, and 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; L3 28I 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 S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E, and S2 39D or S239N; A330L, I332E, and S239D or S239N; V264I, S298A and I332E; S298A, I332E, and 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 K326E or K326T, numbered according to the Kabat EU numbering system. In some embodiments, the cleavable Fc domain is IgG1. The Fc domain or a fragment thereof, and containing one or more amino acid substitutions 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.

[0248] In some embodiments, the cleavable Fc domain further comprises one or more amino acid substitutions that enhance the binding affinity of the half-life-extending domain to FcRn. In some embodiments, one or more amino acid substitutions increase the binding affinity of the Fc-containing polypeptide (e.g., a heavy chain polypeptide or its Fc domain or fragment) to FcRn at acidic pH. In some embodiments, the half-life extension domain comprises one or more amino acid substitutions selected from the following compositions: 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.

[0249] RF mutations or CH3 domain exchanges are used for the purification of heterodimeric proteins. Two immunoglobulin heavy chains differing by at least one amino acid enable the separation of antigen-binding proteins based on the different affinities of the immunoglobulin heavy chains and modified or mutated immunoglobulin heavy chains to affinity reagents. Antigen-binding proteins with IgG CH2 and CH3 regions possessing different affinities for protein A enable rapid separation by the different binding of the IgG region to protein A.

[0250] In one embodiment, the first Fc polypeptide includes a 95R modification (by IMGT exon number; 435R by EU number) in the CH3 region. In another embodiment, the isolated Fc polypeptide further includes a 96F modification (IMGT; 436F by EU number). In some embodiments, the second Fc polypeptide includes wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide includes a 95R / 96F modification by IMGT exon number. In some embodiments, the second Fc polypeptide includes wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the first Fc polypeptide includes a 435R / 436F modification by EU number.

[0251] In some embodiments, the second Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the first Fc polypeptide comprises a CH3 domain derived from IgG3.

[0252] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 33. SEQ ID NO: 33 comprises a "pellet mutation" and an "RF mutation" (435R / 436F).

[0253] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPG (SEQ ID NO: 33) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 33. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 33.

[0254] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 23. SEQ ID NO: 23 comprises a “mortise mutation” of the substrate having SLPLGL (SEQ ID NO: 2).

[0255] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPLGL (SEQ ID NO: 23) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 23. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 23.

[0256] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 24. SEQ ID NO: 24 comprises a “mortise mutation” of the substrate having MPYDLYHP (SEQ ID NO: 5).

[0257] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKMPYDLYHP (SEQ ID NO: 24) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 24. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 24.

[0258] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 25. SEQ ID NO: 25 comprises a “mortise mutation” of the substrate having APAGLIVYN (SEQ ID NO: 7).

[0259] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTAPAGLIVPYN (SEQ ID NO: 25) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 25. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 25.

[0260] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 26. SEQ ID NO: 26 comprises a “mortise mutation” of the substrate having PANLVAPDP (SEQ ID NO: 9).

[0261] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTPANLVAPDP (SEQ ID NO: 26) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 26. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 26.

[0262] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 27. SEQ ID NO: 27 comprises a “mortise mutation” of the substrate having GGPLGL (SEQ ID NO: 3).

[0263] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLGGPLGL (SEQ ID NO: 27) In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 85% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 90% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 91% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 92% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 93% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 94% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 95% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 96% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 97% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 98% identity with SEQ ID NO: 27. In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises an amino acid sequence having at least about 99% identity with SEQ ID NO: 27.

[0264] In some embodiments, the Fc domain comprises a first Fc polypeptide containing the amino acid sequence of SEQ ID NO: 33 and a second Fc polypeptide containing the amino acid sequence of SEQ ID NO: 23.

[0265] In some embodiments, the Fc domain comprises a first Fc polypeptide containing the amino acid sequence of SEQ ID NO: 33 and a second Fc polypeptide containing the amino acid sequence of SEQ ID NO: 24.

[0266] In some embodiments, the Fc domain comprises a first Fc polypeptide containing the amino acid sequence of SEQ ID NO: 33 and a second Fc polypeptide containing the amino acid sequence of SEQ ID NO: 25.

[0267] In some embodiments, the Fc domain comprises a first Fc polypeptide containing the amino acid sequence of SEQ ID NO: 33 and a second Fc polypeptide containing the amino acid sequence of SEQ ID NO: 26.

[0268] In some embodiments, the Fc domain comprises a first Fc polypeptide containing the amino acid sequence of SEQ ID NO: 33 and a second Fc polypeptide containing the amino acid sequence of SEQ ID NO: 27.

[0269] Targeted portion In some embodiments, the masked IL-2 cytokine described herein comprises a targeting moiety (e.g., a targeting cytokine). Therefore, the present invention also provides a targeting cytokine comprising a targeting moiety, a cytokine or a variant thereof (e.g., an IL-2 peptide), a masking moiety (e.g., the anti-IL-2 VHH described herein), and a carrier moiety. In some embodiments, the targeting cytokine comprises a targeting moiety, an IL-2 peptide, a masking moiety, and an Fc domain. The targeting cytokine of the present invention is activated at the disease site and can specifically target target cells, thereby effectively treating cancer without producing unwanted side effects.

[0270] The targeting IL-2 peptide according to this disclosure may bind to the IL-2 peptide or a functional fragment thereof as described anywhere herein; the masking portion as described anywhere herein; the first and second Fc domains as described anywhere herein; the cleavable and non-cleavable linkers as described anywhere herein; and the targeting portion as described anywhere herein.

[0271] This article provides a targeted cytokine comprising a targeting portion. In some embodiments, the targeting portion comprises an antigen-binding portion that binds to an antigen expressed on the surface of a target cell.

[0272] In some embodiments, the targeting portion includes an antigen-binding portion, wherein the antigen is expressed on immune cells. In some embodiments, the targeting portion includes an antigen-binding portion, wherein the antigen is selected from PD-1, PD-L1, CTLA-4, TIGIT, TIM-3, LAG-3, OX40, DR5, ICOS, GITR, CD73, CD39, CD25, CD16a, CD8, KLRC1, KLRD1, KLRB1, CD40, CD137, CD28, and CD16b.

[0273] In some implementations, the target specifically binds to PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CD25, CD16a, CD16b, OX40, DR5, ICOS, GITR, NKG2D, KLRC1, KLRD1, KLRB1, NKP44, NKP30, 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), phosphatidylinositol proteoglycan-3 (GPC3), c-MET, vascular endothelial growth factor receptor 1 (VEGF FR1), vascular endothelial growth factor receptor 2 (VEG FR2), connexin-4, Liv-1, and glycoprotein NMB. (GPNMB), prostate-specific membrane antigen (PSMA), Trop-2, carbonic anhydrase IX (CA9), endothelin B receptor (ETBR), prostate six-span membrane epithelial antigen 1 (STEAPl), NAPI2B, folate receptor α (FR-a), SLIT and NTRK-like protein 6 (SLITRK6), carbonic anhydrase VI (CA6), extracellular nucleotide 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, CD123, CD137, CD138, CD352, CD47, signal regulatory protein α SIRPα, tight junction protein 18.2, tight junction protein 6, 5T4, fibroblast activation protein α (FAPα), fibronectin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or combinations thereof.

[0274] In some embodiments, the targeting portion binds to a tumor-associated antigen. In some embodiments, the targeting portion is an antibody or antigen-binding fragment that binds to a tumor-associated antigen. In some embodiments, the targeting portion is a bispecific antibody or antigen-binding fragment that binds to a tumor-associated antigen. In some embodiments, the targeting portion is an anti-alpha-fetoprotein (AFP) antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-B2M antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-β-human chorionic gonadotropin (β-hCG) antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD117 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD19 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD20 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD22 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD25 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD30 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD33 antibody or a fragment thereof. In some embodiments, the targeting portion is an anti-CD151 antibody or a fragment thereof. In some implementations, the target portion is an anti-MUC-1 antibody or a fragment thereof.

[0275] PD-1 targeting portion In some embodiments, the targeting portion specifically binds to PD-1. In some embodiments, the targeting portion is an anti-PD-1 Fab. In some embodiments, the targeting portion is an anti-PD-1 scFv.

[0276] In some embodiments, the targeting portion is derived from an anti-PD1 antibody. In some embodiments, the targeting portion is derived from pembrolizumab or nivolumab. In some embodiments, the targeting portion is derived from pembrolizumab. In some embodiments, the targeting portion is derived from nivolumab.

[0277] In some implementations, the target portion binds to PD-1.

[0278] In some implementations, the targeting portion includes an agent, peptide, or polypeptide that specifically binds to the target.

[0279] In some embodiments, the targeting portion comprises 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 antibody (bsFab), a single-domain antibody-linked Fab (s-Fab), an antibody, or a combination thereof. In some embodiments, the targeting portion comprises Fab. In some embodiments, the targeting portion comprises a single-chain Fv (scFv). In some embodiments, the targeting portion comprises a single-domain antibody (VHH). In some embodiments, the targeting portion comprises one or more CDRs. In some embodiments, the targeting portion comprises a variable heavy chain (VH). In some embodiments, the targeting portion comprises a variable light chain (VL). In some embodiments, the targeting portion comprises a Fab-like bispecific antibody (bsFab). In some embodiments, the targeting portion comprises a single-domain antibody-linked Fab (s-Fab). In some embodiments, the targeting portion comprises an antibody or a fragment thereof.

[0280] In some implementations, the targeting portion includes the heavy chain variable region or the light chain variable region of pembrolizumab.

[0281] In some implementations, the targeting portion includes the heavy chain variable region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 41).

[0282] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 41. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO: 41.

[0283] In some implementations, the targeting portion comprises the heavy chain variable region and constant region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 49).

[0284] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 49. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO: 49.

[0285] In some implementations, the targeting portion includes the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 43), the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 44), and the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 45).

[0286] In some implementations, the targeting portion contains a light chain variable region of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTSENLYFQ (SEQ ID NO: 42).

[0287] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 42.

[0288] In some implementations, the targeting portion comprises a light chain of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50).

[0289] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 50.

[0290] In some implementations, the targeting portion includes the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 46), the light chain CDR2 sequence of LAS (SEQ ID NO: 47), and the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 48).

[0291] In some implementations, the target portion includes HCDR1 of SEQ ID NO: 43, HCDR2 of SEQ ID NO: 44, HCDR3 of SEQ ID NO: 45, LCDR1 of SEQ ID NO: 46, LCDR2 of SEQ ID NO: 47, and LCDR3 of SEQ ID NO: 48.

[0292] In some implementations, the targeting portion includes the heavy chain variable region or the light chain variable region of nivolumab.

[0293] In some implementations, the targeting portion includes the heavy chain variable region of QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 51).

[0294] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 51.

[0295] In some implementations, the targeting portion includes the following heavy chains: QVQLVESGGG VVQPGRSLRL DCKASGITFS NSGMHWVRQA PGKGLEWVAV IWYDGSKRYYADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATND DYWGQGTLVT VSSASTKGPS VFPLAPCSRSTSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TKTYTCNVDHKPSNTKVDKR VESKYGPPCP PCPAPEFLGG PSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNWYVDGVEVHNA KTKPREEQFN STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRWQEGNVFSCSV MHEALHNHYT QKSLSLSLGK (SEQ ID NO: 52).

[0296] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 52.

[0297] In some implementations, the targeting portion includes the heavy chain CDR1 sequence of GITFSNSG (SEQ ID NO: 53), the heavy chain CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 54), and the heavy chain CDR3 sequence of ATNDDY (SEQ ID NO: 55).

[0298] In some implementations, the targeting portion contains a light chain variable region of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 56).

[0299] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 56. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 56.

[0300] In some implementations, the targeting portion comprises a light chain of EIVLTQSPAT LSLSPGERAT LSCRASQSVSSYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQQ SSNWPRTFGQGTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKDSTYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGE (SEQ ID NO: 57).

[0301] In some embodiments, the targeting portion comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 57. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 57.

[0302] In some implementations, the targeting portion includes the light chain CDR1 sequence of QSVSSY (SEQ ID NO: 58), the light chain CDR2 sequence of DAS (SEQ ID NO: 59), and the light chain CDR3 sequence of QQSSNWPRT (SEQ ID NO: 60).

[0303] In some embodiments, the targeting portion includes the heavy chain CDR1 sequence of GITFSNSG (SEQ ID NO: 53), the heavy chain CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 54), the heavy chain CDR3 sequence of ATNDDY (SEQ ID NO: 55), the light chain CDR1 sequence of QSVSSY (SEQ ID NO: 58), the light chain CDR2 sequence of DAS (SEQ ID NO: 59), and the light chain CDR3 sequence of QQSSNWPRT (SEQ ID NO: 60).

[0304] In some embodiments, the targeting domain is fused to the Fc peptide. In some embodiments, the C-terminus of the targeting domain is fused to the N-terminus of the Fc peptide. In some embodiments, the heavy chain of the Fab is fused to the Fc peptide. In some embodiments, the C-terminus of the heavy chain of the Fab is fused to the N-terminus of the Fc peptide. In some embodiments, the Fc peptide includes a cleavage site.

[0305] In some embodiments, the targeting portion includes a heavy chain variable region, a light chain variable region, and a CH1 domain. In some embodiments, the CH1 domain is derived from IgG1, IgG2, IgG3, or IgG4. In some embodiments, the CH1 domain is derived from IgG1. In some embodiments, the CH1 domain is derived from IgG4.

[0306] Exemplary masking cytokines The present invention provides, in particular, a masked cytokine comprising an IL-2 polypeptide or a variant thereof, a masking moiety, and an engineered Fc domain. The masked IL-2 cytokine of the present invention becomes active at a tumor site by cleaving a cleavage peptide located within the Fc domain of the masked cytokine using a tumor-specific protease, the cleavage peptide releasing the IL-2 polypeptide upon cleavage.

[0307] According to this disclosure, the anti-IL-2 VHH antibody described herein can be used for any masked IL-2 cytokine. In some embodiments, the masked cytokine comprises a carrier portion, a masking portion, and a cytokine. In some embodiments, the masked cytokine comprises a carrier portion, an IL-2 polypeptide as a cytokine, and an anti-IL-2 VHH antibody as the masking portion.

[0308] Specifically, a masked cytokine or targeting cytokine containing the VHH masking portion of the present invention is characterized by: (1) effective masking efficiency, such that the function of IL-2 cytokine is inhibited in undesired targets; (2) efficient activation of IL-2 by protease to release the VHH masking portion; (3) selective activation of IL-2 in tumors rather than in plasma; and (4) in vivo efficacy (e.g., high tumor growth inhibition).

[0309] In some implementations, the masked cytokine comprises an IL-2 peptide, a VHH masking moiety, an anti-PD1 targeting moiety, and an engineered Fc domain containing a tumor-associated protease cleavage site.

[0310] In some implementations, the masked cytokine comprises an IL-2 peptide, a masking portion, a targeting portion, and an engineered Fc domain contained in a tumor-associated protease cleavage site located between EU numbers 438 and 447.

[0311] In some implementations, the masked cytokine comprises a weakened IL-2 peptide, a masking portion, a targeting portion, and an engineered Fc domain contained in a tumor-associated protease cleavage site located between EU numbers 438 and 447.

[0312] In some embodiments, the masked cytokine comprises: a weakened IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); an anti-PD1 targeting portion; and an engineered Fc domain comprising amino acid substitutions of S442G, L443G, S444P, P445L, and G447L.

[0313] In some embodiments, the masked cytokine comprises: a weakened IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); an anti-PD1 targeting portion; and an engineered Fc domain comprising amino acid substitutions of S444P, P445L, and G447L.

[0314] In some implementations, the masked cytokine includes any of the constructs shown in Table D.

[0315] Table D. Exemplary masking cytokines of the present invention In some embodiments, the masked cytokine comprises: an IL-2 polypeptide containing an amino acid substitution of C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and QHSRDLPLT (SEQ ID NO: 48). 48) The anti-PD1 targeting portion of LCDR3; and the engineered Fc domain containing amino acid substitutions of S444P, P445L and G447L.

[0316] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48) and the engineered Fc domain containing amino acid substitutions of S444P, P445L and G447L.

[0317] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and QHSRDLPLT (SEQ ID NO: 48). 48) The anti-PD1 targeting portion of LCDR3; and the engineered Fc domain containing amino acid substitutions of S444P, P445L and G447L.

[0318] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48) and the engineered Fc domain comprising amino acid substitutions of S440M, L441P, S442Y, L443D, S444L, P445Y, G446H and G447P.

[0319] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48); and engineered Fc domains comprising amino acid substitutions of Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N.

[0320] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48) and engineered Fc domains comprising amino acid substitutions of Q438P, K439A, S440N, S442V, L443A, S444P, P445D and G446P.

[0321] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48) and engineered Fc domains containing amino acid substitutions S442G, L443G, S444P, P445L and G447L.

[0322] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of R38A, F42A, Y45A, E62A, and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and CDR3 of sequence QHSRDLPLT (SEQ ID NO: 48). The anti-PD1 targeting portion of LCDR3 (NO:48) and the engineered Fc domain containing amino acid substitutions S444P, P445L and G447L.

[0323] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSFYEDETDY (SEQ ID NO: 17); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and QHSRDLPLT (SEQ ID NO: 48). 48) The anti-PD1 targeting portion of LCDR3; and engineered Fc domains containing amino acid substitutions S444P, P445L and G447L.

[0324] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSFYEDETDY (SEQ ID NO: 17); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and QHSRDLPLT (SEQ ID NO: 48). 48) The anti-PD1 targeting portion of LCDR3; and engineered Fc domains comprising amino acid substitutions S442G, L443G, S444P, P445L and G447L.

[0325] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising amino acid substitutions of F42E and C125A; a VHH masking portion comprising CDR1 of sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of sequence ASSWYEDETDY (SEQ ID NO: 16); and HCDR1 of sequence GYTFTNYY (SEQ ID NO: 43), HCDR2 of sequence INPSNGGT (SEQ ID NO: 44), HCDR3 of sequence ARRDYRFDMGFDY (SEQ ID NO: 45), LCDR1 of sequence KGVSTSGYSY (SEQ ID NO: 46), LCDR2 of sequence LAS (SEQ ID NO: 47), and QHSRDLPLT (SEQ ID NO: 48). 48) The anti-PD1 targeting portion of LCDR3; and engineered Fc domains comprising amino acid substitutions S442G, L443G, S444P, P445L and G447L.

[0326] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 11; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23.

[0327] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23.

[0328] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23.

[0329] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 24.

[0330] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 25.

[0331] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 26.

[0332] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27.

[0333] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 20; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23.

[0334] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 20; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27.

[0335] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27.

[0336] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 21; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27.

[0337] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 11; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0338] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0339] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0340] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 24, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0341] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 25, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0342] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 26, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0343] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 12; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0344] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 20; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 23, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0345] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 20; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and an engineered Fc domain comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0346] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 19; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0347] In some embodiments, the masked cytokine comprises: an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 13; a VHH masking portion comprising the amino acid sequence of SEQ ID NO: 21; an anti-PD1 targeting portion comprising the amino acid sequences of SEQ ID NO: 41 and 42; and engineered Fc domains comprising a first Fc polypeptide of SEQ ID NO: 33 and a second Fc polypeptide of SEQ ID NO: 27, wherein the IL-2 polypeptide is linked to the first Fc polypeptide and the VHH masking portion is linked to the second Fc polypeptide.

[0348] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63, SEQ ID NO: 64, or SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, or SEQ ID NO: 74; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0349] In some embodiments, the IL-2 peptide is fused to the first Fc peptide via a GS linker. In some embodiments, the IL-2 peptide is fused to the first Fc peptide via a GGGGS (SEQ ID NO: 61) linker. In some embodiments, the IL-2 peptide is fused to the first Fc peptide via a GGGGSGGGGSGGGGS (SEQ ID NO: 62) linker.

[0350] In some embodiments, the VHH masking portion is fused to the second Fc peptide via a GS linker. In some embodiments, the VHH masking portion is fused to the second Fc peptide via a GGGGS (SEQ ID NO: 61) linker. In some embodiments, the VHH masking portion is fused to the second Fc peptide via a GGGGSGGGGSGGGGS (SEQ ID NO: 62) linker.

[0351] SEQ ID NO: 63 comprises, from the N-terminus to the C-terminus, a variable heavy chain that binds to PD1, an uncleavable Fc peptide, and attenuated IL-2. (SEQ ID NO: 63) SEQ ID NO: 64 contains, from the N-terminus to the C-terminus, a variable heavy chain that binds to PD1, an uncleavable Fc polypeptide, and wild-type IL-2. QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGSSPPGGGSSGGGSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO: 64) SEQ ID NO: 65 comprises, from N-terminus to C-terminus, a PD1-binding variable heavy chain, a non-cleavable Fc polypeptide, and non-alpha IL-2: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGGSSPPGGGSSGGGSGPAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTAKFAMPKKATELKHLQCLEEALKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT(SEQ ID NO: 65) From the N-terminus to the C-terminus, SEQ ID NO: 66 comprises a PD1-binding variable heavy chain, an Fc polypeptide comprising the cleavable substrate SLPLGL (SEQ ID NO: 2), and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPLGLGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 66) SEQ ID NO: 67 comprises, from the N-terminus to the C-terminus, a PD1-binding variable heavy chain, an Fc polypeptide comprising a cleavable substrate of GGPLGL (SEQ ID NO: 3), and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLGGPLGLGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 67) SEQ ID NO: 68 comprises, from N-terminus to C-terminus, a PD1-binding variable heavy chain, an Fc polypeptide comprising a cleavable substrate MPYDLYHP (SEQ ID NO: 5) and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKMPYDLYHPGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 68) SEQ ID NO: 69 comprises, from N-terminus to C-terminus, a PD1-binding variable heavy chain, an Fc polypeptide comprising the cleavable substrate APAGLIVPYN (SEQ ID NO: 7), and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTAPAGLIVPYNGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 69) SEQ ID NO: 70 comprises, from N-terminus to C-terminus, a variable heavy chain that binds PD1, an Fc polypeptide comprising a cleavable substrate of PANLVAPDP (SEQ ID NO: 9) and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTPANLVAPDPGGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 70) SEQ ID NO: 71 comprises, from N-terminus to C-terminus, a PD1-binding variable heavy chain, an Fc polypeptide comprising a cleavable substrate SLPLGL (SEQ ID NO: 2) and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPLGLGGSSGSGGSGGGSGSGGGSGGSGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 71) SEQ ID NO: 72 comprises, from the N-terminus to the C-terminus, a PD1-binding variable heavy chain, an Fc polypeptide comprising a cleavable substrate of SLPLGL (SEQ ID NO: 2) and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPLGLGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKGRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAYASSYYEDETDYWGQGTQVTVSS (SEQ ID NO: 72) From the N-terminus to the C-terminus, SEQ ID NO: 73 comprises a PD1-binding variable heavy chain, an Fc polypeptide comprising a cleavable substrate GGPLGL (SEQ ID NO: 3) and a VHH masking moiety: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLGGPLGLGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKGRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAYASSYYEDETDYWGQGTQVTVSS (SEQ ID NO:73) SEQ ID NO: 74 comprises, from the N-terminus to the C-terminus, a variable heavy chain that binds to PD1, an Fc polypeptide containing a substrate cleavable by GGPLGL (SEQ ID NO: 3), and a VHH masking moiety with an alanine extension. QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLGGPLGLGGSSGSGGSGGGSGSGGGEVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSSAAA (SEQ ID NO: 74) SEQ ID NO: 75 comprises a variable heavy chain that binds PD1 and an Fc polypeptide from the N-terminus to the C-terminus: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQG TTVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 75) In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 64; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0352] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0353] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 66; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0354] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 68; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0355] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 69; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0356] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 70; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0357] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 67; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0358] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 71; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0359] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 72; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0360] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 73; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0361] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 63; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 67; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0362] In some embodiments, the masked cytokine comprises: a first polypeptide comprising the amino acid sequence of SEQ ID NO: 65; a second polypeptide comprising the amino acid sequence of SEQ ID NO: 74; and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 50.

[0363] Example Although certain compounds, compositions and methods of the present invention have been specifically described according to some embodiments, the following examples are only used to illustrate the compounds of the present invention and are not intended to limit the scope of the compounds.

[0364] Example 1. The IL-2 cytokine in the masked cytokine construct is effectively masked by the VHH masking component.

[0365] This embodiment illustrates that masked IL-2 cytokines with cleavable Fc domains are effectively masked by the VHH masking portion and can be successfully cleaved by tumor-specific proteases, thereby restoring the potency of IL-2 cytokines.

[0366] Various masked and IL-2-targeting cytokines as shown in Table 1 were prepared. Each masked and targeted cytokine consists of three chains: chain 1 contains PD-1 targeting the VH+ uncleavable Fc domain + IL-2 cytokine; chain 2 contains PD-1 targeting the VH+ cleavable Fc domain + VHH masking fragment; and chain 3 contains PD-1 targeting VL. Figure 1 An exemplary construct is shown. Recombinant human IL-2 (rhIL-2) and a control UCM without the masking component are used as controls.

[0367] Table 1. Exemplary targeting and masking IL-2 cytokines with cleavable Fc domains The percentage of STAT5 phosphorylation was measured to determine the effectiveness of the VHH-masked portion in masking the IL-2 cytokine. Briefly, pre-activated primary human peripheral blood mononuclear cells (PBMCs) upregulated PD-1 expression. Pre-activated PBMCs were incubated with exemplary constructs at various doses listed in Table 1 for 12 minutes, followed by evaluation of STAT5 phosphorylation. Constructs with the VHH-masked portion exhibited minimal activity against hPBMCs, while the unmasked control constructs showed potent signaling against CD8+ T cells. Results for UCM control 1, UCM3, UCM9, and UCM11 are as follows: Figure 2A As shown, and the results of UCM controls 2 and 3, and UCM1 and UCM2 are as follows: Figure 2B As shown.

[0368] Example 2. In vitro cleavage of masked IL-2 cytokines with cleavable Fc domains This embodiment illustrates that masked IL-2 cytokines containing cleavable Fc domains can be cleaved by human tumors, but not by plasma.

[0369] Frozen cells extracted from fresh tumor tissue (X-FACT assay) and human plasma were used for the ex vivo cleavage of masked cytokines with cleavable Fc domains. Cells from five different indications—melanoma, HNC (head and neck cancer), lung cancer, colon cancer, and renal cell carcinoma (RCC)—were used for the cleavage assay. UCM2, UCM3, UCM4, UCM5, UCM6, UCM7, UCM8, UCM9, and UCM11 (as shown in Table 1), along with non-cleavable control molecules, were incubated with frozen tumor cells or in human plasma derived from healthy donors and patients with melanoma, HNC, lung cancer, colon cancer, and RCC.

[0370] The cleavage results were analyzed using Western blotting. Results for UCM3, UCM9, and UCM9 are as follows: Figure 3A and Figure 3B As shown, the results for UCM2, UCM3, UCM4, UCM5, UCM6, UCM7, and UCM8 are as follows: Figure 3C and Figure 3D As shown. Figure 3A and Figure 3C As shown, all masked IL-2 cytokines were cleaved by human tumor cells in vitro. On the other hand, masked cytokines were not cleaved by human plasma, indicating that they were cleaved as desired for tumor-specific cleavage. Figure 3B and Figure 3D ).

[0371] Next, fresh tumor cells from five different indications (HNC (head and neck cancer), lung cancer, colon cancer, renal cell carcinoma (RCC), and sarcoma) were used for dissection assays. UCM11 was tested and compared with an undissectable control. Figure 3E As shown, UCM11 exhibited a significantly higher cutting percentage compared to the indivisible control. The indivisible control had a cutting percentage of less than approximately 3%, while UCM11 had a cutting percentage of at least greater than 4% and up to 13.4%, indicating significant cutting.

[0372] Next, fresh tumor cells from three different indications (HNC (head and neck cancer), lung cancer, and renal cell carcinoma (RCC)) were used for cleavage assays. UCM12 was tested and compared with an uncleavable control. Figure 3F As shown, UCM12 exhibited a significantly higher cut rate compared to the uncut control. The uncut control had a cut rate of less than 1%, while UCM12 had a cut rate as high as 4.7%, consistent with the tumor-mediated cut rate of UCM12.

[0373] Example 3. In vivo efficacy of masked cytokines with Fc domain cleavage capabilities This example illustrates that both targeted and masked IL-2 cytokines containing cleavable Fc domains are effective in vivo. In this specific study, human PD-1 transgenic mice carrying mouse tumors were used.

[0374] In one study, C57BL / 6-B-hPD1 mice were subcutaneously implanted with MC38 tumor cells and received two intravenous injections of 8 mg / kg UCM3, UCM9, or UCM11 (N=8) and 0.75 mg / kg control UCM1 (N=8) or a carrier (N=8). Tumor and body weight were measured two or three times per week.

[0375] The results are as follows Figure 4A and Figure 4B As shown. Overall, the data indicate that the masked portion of IL-2-targeting cytokines with engineered Fc cleavable domains... exist It is effectively cleaved in vivo, thereby activating IL-2 activity. Compared with mediators, the activated targeting cytokine can effectively inhibit tumor growth. Figure 4A In addition, the overall survival rate of treated mice increased ( ). Figure 4B ).

[0376] In another study, the same methodology was used to evaluate the in vivo efficacy of UCM2. Control UCM3 and a reference PD-1 antibody were used as controls. Overall, the data suggest that the masked portion of the IL-2-targeting cytokine, engineered with an Fc-cleavable domain, is effective. exist It is effectively cleaved in vivo, thereby activating IL-2 activity. Compared with mediators, the activated targeting cytokine can effectively inhibit tumor growth. Figure 4C In addition, the overall survival rate of treated mice increased ( ). Figure 4D ).

[0377] In another study, the in vivo efficacy of UCM12 in the bladder mouse model MB49 was also evaluated using the same methods described herein. Specifically, MB49 tumor cells were subcutaneously implanted in C57BL / 6-hPD1 mice, which then received two intravenous injections of 10 or 3 mg / kg UCM12 (N=9) or the carrier (N=9). An equimolar dose of pembrolizumab (N=9) was also used as a control. Tumor and body weight were measured two or three times weekly.

[0378] The results of UCM12 are as follows Figure 4E and Figure 4F As shown. Overall, the data indicate that the masked portion of IL-2-targeting cytokines with engineered Fc cleavable domains... exist It is effectively cleaved in vivo, thereby activating IL-2 activity. Compared with mediators, the activated targeting cytokine can effectively inhibit tumor growth. Figure 4E In addition, the overall survival rate of treated mice increased ( ). Figure 4F ).

[0379] Example 4. Validation of the in vivo efficacy of masked cytokines with Fc domain cleavage capabilities. This embodiment verifies that targeted and masked IL-2 cytokines containing cleavable Fc domains are effective in vivo. efficient of. In this study, human PD-1 transgenic mice carrying mouse tumors were used.

[0380] C57BL / 6-hPD1 mice were subcutaneously implanted with MC38 tumor cells and received two intravenous injections of 7.5 mg / kg UCM4, UCM6, UCM7, or UCM8 (N=8) or a carrier (N=8). Tumor size and body weight were measured two or three times per week.

[0381] The results are as follows Figure 5A and Figure 5B As shown. Overall, the data indicate that the masked portion of IL-2-targeting cytokines with engineered Fc cleavable domains... exist It is effectively cleaved in vivo, thereby activating IL-2 activity. Compared with mediators, the activated targeting cytokine can effectively inhibit tumor growth. Figure 5A Additionally, mice treated with UCM4 or UCM7 showed an increased overall survival rate. Figure 5B ).

[0382] Example 5. In the masked cytokine construct, IL-2 cytokine is effectively masked by the VHH masking component. It is activated by tumor-associated proteases.

[0383] The in vitro cleavage of masked cytokines with cleavable Fc domains by tumor-associated proteases was tested. In this specific study, UCM12 was cleaved by MMP9. The protease was activated by diluting recombinant human protease MMP-9 to 0.22 mg / mL in MMP buffer (150 mM NaCl; 50 mM Tris, pH 7.5; 10 mM CaCl2) and incubating it with an equal volume of 1 mM 4-aminophenylacetic acid mercuric acid at 37°C for 24 h. Subsequently, each sample was diluted to 1 μM in MMP buffer, and two aliquots were prepared: 100 μL (cleaved) and 300 μL (uncleaved). 25 ng of pre-activated MMP9 was added to the cleavage tube. The same volume of buffer was added to the uncleaved tube. The samples were incubated at 37°C for 18 h. Cleavage was confirmed by SDS-PAGE under reducing and denaturing conditions.

[0384] like Figure 6AThe SDS-PAGE image shown indicates that UCM12 was effectively cut by MMP9, producing the same control band as the control UCM1 (unmasked control).

[0385] In addition to cleavage by MMP9, in vitro proteolytic cleavage of various other tumor-associated proteases possessing Fc domain-cleavable cytokines was tested. To measure the catalytic efficiency of human proteases in UCM12 cleavage, UCM12 was incubated with matrix metalloproteinase 1 (MMP1), MMP2, MMP8, MMP9, or cathepsin L. Aliquots were taken during the reaction and analyzed by capillary electrophoresis-sodium dodecyl sulfate. The amount of UCM12 cleaved was determined using LabChip GX Reviewer software. Figure 6C and 6D The data in Table 5 show that UCM12 can be effectively cleaved and activated by a variety of proteases.

[0386] Table 5: Catalytic efficiency of the tested proteases for UCM12 cleavage Next, the percentage of STAT5 phosphorylation was measured to determine the effectiveness of the VHH masking moiety in masking IL-2 cytokines and its activation via tumor-associated cleavage.

[0387] Matrix metalloproteinase-9 (MMP9) was prepared in MMP buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5) with 1 mM right Mercuric aminophenylacetate was activated overnight at 1.3 μM. The next day, the test sample (2 μM) was cleaved overnight at 37°C in MMP buffer with shaking using 25 nM pre-activated MMP9. The cleavage was confirmed using an SDS-PAGE.

[0388] pSTAT5 activity (STAT5 phosphorylation) was measured on human peripheral blood mononuclear cells (PBMCs).

[0389] In short, 96-well plates were coated with 1 μg / mL anti-CD3 antibody (clone: ​​OKT3) in phosphate-buffered saline (PBS) and incubated at 37°C for 1 hour. Simultaneously, frozen hPBMCs were thawed in a 37°C water bath and then transferred to complete culture medium (Roswell Park Memorial Institute 1640 medium [RPMI-1640], 10% fetal bovine serum [FBS], 50 U / mL penicillin, 50 μg / mL streptomycin, 2 mM L-glutamine, 1 μM each of the following non-essential amino acids (glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine), 10 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, and 10 μg / mL gentamicin). hPBMCs were then counted, centrifuged at 450g for 4 min, the supernatant was discarded, and cells were transferred at 2 x 10⁻⁶ cells / mL. 6 hPBMCs were resuspended at 100 cells / mL in complete culture medium. The CD3-coated 96-well plates were washed three times with PBS. Anti-CD28 antibody (clone: ​​CD28.2) was added to the hPBMCs at a final concentration of 1 μg / mL, and 200 μL of hPBMC cell suspension was added to each well of the CD3-coated 96-well plates. The plates were incubated at 37°C for two days. Subsequently, hPBMCs were collected and counted. The hPBMCs were washed twice with complete culture medium and then resuspended at 2 x 10⁻⁶ cells / mL in complete culture medium. 6 / mL resuspend. 100 μL of hPBMC was seeded again in each well of a 96-well round-bottom plate and incubated overnight at 37°C.

[0390] The next day, serial dilutions of the test product were diluted to twice the final concentration in complete culture medium. The plate was removed from the incubator, and 50 μL was carefully dispensed from each well. An equal volume (50 μL) of the construct was added to 50 μL of cells and incubated at 37°C. After 12 minutes, paraformaldehyde was added directly to the cells to a final concentration of 2.7%, and then incubated at 37°C for 15 minutes.

[0391] Cells were then washed once with 200 μL of Fluorescence Activated Cell Sorting (FACS) buffer (1xPBS, 2% FBS). Cells were resuspended in 200 μL of Perm III buffer (BD Biosciences) and incubated on ice for 30 min. Cells were washed three times with 200 μL of FACS buffer and then resuspended in 100 μL of antibody mixture in FACS buffer. Cells were incubated on ice in the dark for 30 min. An additional 150 μL of FACS buffer was added to each well and centrifuged at 450 g for 4 min. The plate was washed again with 200 μL of FACS buffer. The stained hPBMCs were resuspended in 150 μL of FACS and run on a BDFortessa cytometer.

[0392] Figure 6B The data presented in Table 2 show that UCM11 and UCM12 are effectively masked in the absence of cleavage; MMP9-cleaved UCM11 and UCM12 exhibit enhanced pSTAT5 activity. Table 2 also shows that the EC50 values ​​of UCM11 and UCM12 decreased significantly (at least 300-fold) upon the addition of MMP9, indicating increased potency after protein hydrolysis activation.

[0393] Table 2. EC5 values ​​of masked IL-2 cytokines with or without MMP9 Example 6. Verification of effective masking by VHH and restoration of IL-2 activity by proteolytic cleavage This embodiment verifies that masked IL-2 cytokines with cleavable Fc domains are effectively masked by the VHH masking portion and can be successfully cleaved by tumor-specific proteases, thereby restoring the potency of IL-2 cytokines.

[0394] HEK Blue IL-2 cells (Invivogen) were grown as follows: In short, cells were passaged in growth medium (DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% FBS, 50 U / mL penicillin, 50 μg / mL streptomycin, 100 μg / mL streptomycin, 1x HEK-Blue CLR selection agent, 1 μg / mL puromycin) until approximately 80% confluence. Cells were then centrifuged at 300g for 5 minutes and reconstituted with 10% DSM-SO, 20% fetal bovine serum, and 70% DMEM, 4.5 g / L glucose, and 2 mM L-glutamine. They were then frozen at -80°C for 12 hours in Corning CoolCell freezers and subsequently stored in liquid nitrogen for long-term preservation to establish a "ready-to-use" cell bank. For the assay, frozen "ready-to-use" HEK BlueIL-2 cells were rapidly thawed in a 37°C water bath and then transferred to 50 mL conical tubes containing assay medium (DMEM, 4.5 g / L glucose, 2 mL glutamine, 10% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin). The cells were centrifuged at 300 g for 5 minutes and then thawed with assay medium at 0.3 x 10⁻⁶ cells / mL. 6 Cells / mL remodeling. Cells were seeded at 50,000 cells per well in 96-well plates. 50 μL aliquots of serially diluted test samples in the assay medium were added to the cells, with the final concentration of the uncut test sample ranging from 1.2 to 100,000 pM and the final concentration of the cut test sample ranging from 0.1 to 8750 pM. The plates were incubated at 37°C and 5% CO2 for 24 hours.

[0395] 24 hours later, Quanti-Blue solution (Invivogen) was prepared according to the manufacturer's recommendations. 20 μL of cell supernatant was added to 180 μL of Quanti-Blue solution in a fresh 96-well plate. The plate was sealed, gently tapped to mix, and incubated at 37°C for 2 hours. Afterward, the seal was removed, and absorbance was measured at 625 nm.

[0396] Data analysis was performed using Graphpad Prism version 10.1.2. Background (assay medium) was subtracted from the raw data to establish a reference subtracted light units (RSLU), and the data were fitted to a nonlinear regression equation: [agonist] to response – variable slope (four parameters). EC50 for each construct is reported. 50 value.

[0397] Figure 7The data presented indicate that UCM10, UCM11, and UCM12 were effectively masked in the absence of cleavage; after cleavage by MMP9, UCM10, UCM11, and UCM12 exhibited similar activity to rhIL2, indicating successful unmasking.

[0398] Example 7. Exemplary masked IL-2 cytokine blocking PD-1 / PD-L1 interaction This embodiment demonstrates that exemplary masked IL-2 cytokines maintain the ability to target PD-1 and block the PD-1 / PD-L1 axis.

[0399] The ability of an exemplary masking IL-2 cytokine and a reference anti-PD-1 antibody to target PD-1 was tested using an ELISA binding assay. ELISA plates were coated with recombinant human PD-1, followed by blocking at concentrations ranging from 30 to 0.0003 nM and incubation with the test product. Immobilized test products were detected colorimetrically using a horseradish peroxidase (HRP)-conjugated polyclonal anti-human fragment crystallized antibody. Data indicate that the tested exemplary masking IL-2 cytokine and reference PD-1 antibody are equivalent binders to human PD-1.

[0400] Next, exemplary masked IL-2 cytokines and a reference anti-PD1 antibody with PD-1 blocking activity were tested using the Promega PD-1 / PD-L1 blocking bioassay. The bioassay was set up and run according to the manufacturer's protocol.

[0401] In short, prepare the cell recovery medium provided with the bioassay kit one day prior to the assay by adding the provided FBS (90% Ham's F-12 / 10% FBS). Gently thaw the PD-L1 aAPC / CHO-K1 cell vials in a 37°C water bath for 3 minutes. Add the thawed PD-L1 aAPC / CHO-K1 cell vials to the preheated recovery medium and mix the cell solution by inverting the plate twice. Add 100 μL of the cell suspension to a 96-well white flat-bottomed assay plate treated with tissue culture. Cap the plate and incubate overnight (37°C, 5% CO2).

[0402] On the second day, remove the assay plate containing PD-L1 aAPC / CHO-K1 cells from the incubator and remove 95 μL of recovery medium from each well. Prepare serial dilutions of the test products (exemplary masking IL-2 cytokine and reference antibody) in the provided assay buffer (99% RPMI 1640 / 1% FBS). Add 40 μL aliquots of the serial dilutions to the wells containing PD-L1 aAPC / CHO-K1 cells, where the final concentration of the test products is in the range of 0.033–50 nM. Next, prepare the cells by thawing the PD-1 effector cell vials in a 37°C water bath for 3 minutes and adding the thawed effector cells to the pre-warmed assay buffer. Mix the cell suspension by inverting twice, then add 40 μL of the effector cell suspension to each well. Cap the assay plate and incubate for 6 hours (37°C, 5% CO2). After 6 hours of incubation, remove the assay plate from the incubator and allow it to equilibrate at ambient temperature for 5–10 minutes. Add 80 μL of Bio-Glo reagent to each well and incubate the plate at ambient temperature for 5 minutes before measuring the luminescence.

[0403] In this assay, an exemplary masking IL-2 cytokine was added to block the interaction between PD-L1 from PD-L1 aAPC / CHO-K1 and PD-1 from PD-1 effector cells. By blocking this interaction, the exemplary masking IL-2 cytokine releases an inhibitory signal, thereby inducing T cell receptor (TCR) signaling and resulting in increased luminescence. Figure 8 The fold induction of luminescence in an exemplary masked IL-2 cytokine is shown in an exemplary experiment.

[0404] Exemplary masked IL-2 cytokines were tested using a bioassay based on Promega PD-1 / PD-L1 blocking cells. As discussed above, the assays were set up and run according to the manufacturer's protocol, with the final concentrations of the test products (i.e., the exemplary masked IL-2 cytokines and the reference anti-PD1 antibody) ranging from 0.033 to 50 nM. Data analysis was performed in GraphPadPrism. Fold-induction was calculated by subtracting the background luminescence from the luminescence of the test product and then dividing by the product of the antibody-free control and the background (RLU[antibody – background] / RLU[antibody-free control – background]). Table 3 shows the calculations for each exemplary masked IL-2 cytokine. EC50 Values. The data presented in Table 3 show that all exemplary masked IL2 cytokines tested exhibited comparable ability to block the PD-1 / PD-L1 axis to the reference anti-PD1 antibody.

[0405] Table 3. EC50 values ​​of exemplary masked IL-2 cytokines In summary, this embodiment demonstrates that the exemplary masked IL-2 cytokine has a similar ability to block the PD-1 / PD-L1 axis as a reference anti-PD1 antibody with known blocking activity.

[0406] Example 8. Effective elimination of the combination of VHH masking and CD122. This embodiment verifies that the exemplary masked IL-2 cytokine UCM12 can be effectively masked by the VHH masking component. Surface plasmon resonance (SPR) measurements of the binding affinity of UCM12 to CD25 and CD122 were performed and compared with the binding affinity of unmasked controls (control UCM1 and control UCM2) and recombinant human IL-2 (rhIL-2; AcroBiosystems).

[0407] SPR measurements were performed at 25°C using a BIAcore T200 system equipped with the Biotin Capture Kit S Series (Cytiva). HBS-EP+ buffer (150 mM NaCl; 10 mM HEPES; 3 mM EDTA and 0.05% (v / v) surfactant P20 pH 7.4) was used as the run buffer. The chip was prepared according to the supplier's recommendations. Briefly, the chip was hydrated and surface-treated by injecting a regeneration solution (6M guanidine-HCl; 0.25M NaOH) three times, one minute each time. Three start-up cycles were then run by injecting the biotin capture reagent (Cytiva), ligand, analyte, and regeneration solution.

[0408] At the start of each cycle, a biotin-capturing reagent was applied to the chip for 5 min. Biotinylated recombinant human CD25 or CD122 (AcroBiosystems) was used as the ligand. The ligand was diluted in run buffer to obtain 0.04–2.0 μg / ml ligands and immobilized on flow channel 2 of the chip. Flow channel 1 was left empty as a negative control. The test products were serially tripled in run buffer to obtain test products ranging from 18.52 to 13,500 nM. Next, they were applied to the chip surface and flowed for 2 min. The molecules were then allowed to dissociate for 5 min, followed by regeneration with regeneration solution for 2 min after each cycle. To measure binding affinity, steady-state affinity analysis was performed using the empirical BIACORE T200 evaluation software version 3.2.1.

[0409] As shown in Table 4, the exemplary masked IL-2 cytokine UCM12 was effectively masked because it did not bind to CD122, unlike the unmasked control. Furthermore, UCM12 showed a 236-fold reduced affinity for CD25 compared to rhIL-2.

[0410] Table 4. Binding affinity of exemplary masked IL-2 cytokine UCM12 to human CD25 or CD122 Example 9. Exemplary masked IL-2 cytokines exhibit antibody-like pharmacokinetics This embodiment demonstrates the antibody-like pharmacokinetics of the exemplary masked IL-2 cytokine UCM12 in a mouse model.

[0411] Nonclinical pharmacokinetics (PK) of exemplary masked IL-2 cytokines, UCM12, and unmasked UCM control 1 were evaluated in human neonatal fragment crystallization (FcRn) mice. Mice were administered intravenous (IV) infusions of single doses of 1 mg / kg, 3 mg / kg, or 10 mg / kg of UCM12 or equimolar doses of UCM control 1.

[0412] Animals were bled at 1, 6, 24, 72, 120, 168, 240, and 336 hours post-drug administration for PK analysis. Whole blood (approximately 50 μL) was collected via submandibular bleeding into heparin-coated tubes and immediately inverted multiple times. Plasma was separated by centrifugation at 1,000 to 2,000 × g for 10 minutes at 4°C. Immediately after centrifugation, approximately 25 μL of the resulting supernatant (plasma) was transferred to designated tubes and stored at -80°C. Plasma samples were maintained at 2°C to 8°C during processing.

[0413] Plasma levels of UCM12 and UCM control 1 were measured using Mesoscale Discovery (MSD) assays with anti-human Fc capture antibody and anti-human Fc detection antibody. Pharmacokinetic parameters of total UCM12 and UCM control 1 in plasma were determined using non-compartmental analysis (NCA) with WinNonlin v8.1 software.

[0414] Figure 9 Plasma levels and PK parameters of UCM12 and control UCM1 are shown. These data indicate that UCM12 exhibits antibody-like PK in the hFcRn mouse model.

[0415] Example 10. Exemplary masked IL-2 cytokines showed reduced proliferation of PBMCs. This embodiment demonstrates that the exemplary masking IL-2 cytokine effectively masks the stimulation of peripheral blood mononuclear cell (PBMC) proliferation.

[0416] Human peripheral blood mononuclear cells (PBMCs) were thawed and pre-activated. The cells were then mixed with a specified concentration (e.g., ...). Figure 10A and 10B The test preparation (including exemplary masked IL-2 cytokines UCM3, UCM9, UCM11, and UCM12, recombinant human IL-2 as a control, and control UCM1) was mixed. After incubation for 4–5 days, cells were harvested and proliferation was measured using Cell Titer Glo. The data presented indicate that UCM3, UCM9, UCM11, and UCM12 had lower activity than recombinant human IL-2 (…). Figure 10A and 10B The data also showed that UCM12, compared to the unmasked control UCM1, exhibited reduced proliferation of human PBMCs. Figure 10B ).

[0417] Equivalent solution Those skilled in the art will recognize or be able to determine, using only conventional experiments, that many embodiments are equivalent to the specific embodiments of the invention described herein. The scope of the invention is not limited to the foregoing description, but rather as set forth in the following claims.

Claims

1. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide, The masking component contains only heavy chain antibodies (VHH). Anti-PD1 targeting component, and An engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. The first Fc polypeptide contains a tumor-associated protease cleavage site and is fused with the IL-2 polypeptide or the masking portion, such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

2. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide, Concealed area, Targeted portion, and An engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. The first Fc polypeptide contains a tumor-associated protease cleavage site located between EU numbers 438 and 447; The first Fc polypeptide is fused with the IL-2 polypeptide or the masking portion, such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

3. A masking cytokine comprising: Attenuated interleukin-2 (IL-2) peptide, Concealed area, Targeted portion, and An engineered Fc domain comprising a first Fc polypeptide and a second Fc polypeptide. The first Fc polypeptide contains a tumor-associated protease cleavage site of the sequence PLGL (SEQ ID NO: 1); The first Fc polypeptide is fused with the IL-2 polypeptide or the masking portion, such that the masking portion binds to the IL-2 polypeptide, and the IL-2 polypeptide is released from the masking portion when the tumor-associated protease cleavage site in the first Fc polypeptide is cleaved.

4. The masking cytokine of any one of claims 2 to 3, wherein the masking portion comprises heavy chain-only antibody (VHH).

5. The masked cytokine according to any one of claims 2 to 4, wherein the targeting portion is an anti-PD1 targeting portion.

6. The masked cytokine of any one of claims 1 to 5, wherein the tumor-associated protease cleavage site is located between EU number positions 444-447, 440-447, 438-447, 438-446, or 442-447.

7. The masked cytokine as claimed in any of the preceding claims, wherein the tumor-associated cleavage site is located between EU numbers 442 and 447.

8. The masked cytokine of any one of claims 1, 2, or 4 to 7, wherein the tumor-associated cleavage site comprises the amino acid sequence of PLGL (SEQ ID NO: 1), MPY (SEQ ID NO: 4), APAG (SEQ ID NO: 6), or PAN (SEQ ID NO: 8).

9. The masked cytokine as claimed in any of the preceding claims, wherein the engineered Fc polypeptide comprises amino acid substitutions of EU numbers S442G, L443G, S444P, P445L and G447L.

10. The masked cytokine of any one of claims 1 to 8, wherein the engineered Fc polypeptide comprises amino acid substitutions of EU numbers S444P, P445L, and G447L.

11. The masked cytokine of any one of claims 1 to 8, wherein the engineered Fc polypeptide comprises amino acid substitutions of EU numbers S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P.

12. The masked cytokine of any one of claims 1 to 8, wherein the engineered Fc polypeptide comprises amino acid substitutions of EU numbers Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N.

13. The masked cytokine of any one of claims 1 to 8, wherein the engineered Fc polypeptide comprises amino acid substitutions of EU numbers Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

14. The masked cytokine as claimed in any of the preceding claims, wherein the IL-2 comprises modifications of R38A, F42A, Y45A and E62A relative to the sequence of mature IL-2 having SEQ ID NO:

10.

15. The masked cytokine of any one of claims 1 to 14, wherein the IL-2 contains a modified C125A sequence relative to the mature IL-2 having SEQ ID NO:

10.

16. The masked cytokine of any one of claims 1 to 13, wherein the IL-2 comprises the modifications F42E and C125A relative to the sequence of mature IL-2 having SEQ ID NO:

10.

17. The masked cytokine of any one of claims 1 to 16, wherein the VHH comprises CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16).

18. The masked cytokine of any one of claims 1 to 16, wherein the VHH comprises CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSFYEDETDY (SEQ ID NO: 17).

19. A masking cytokine comprising: A weakened interleukin-2 (IL-2) polypeptide containing amino acid substitutions for F42E and C125A. The VHH masking portion includes CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16). Anti-PD1 targeting component, and The Fc domain contains a first Fc polypeptide and a second Fc polypeptide. The first Fc polypeptide contains amino acid substitutions of S442G, L443G, S444P, P445L, and G447L to engineered tumor-associated protease cleavage sites, while the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and The first Fc polypeptide is linked to the VHH masking portion and the Fc polypeptide is linked to the attenuated IL-2 polypeptide.

20. A masking cytokine comprising: A weakened interleukin-2 (IL-2) polypeptide containing amino acid substitutions for F42E and C125A. The VHH masking portion includes CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSFYEDETDY (SEQ ID NO: 17). Anti-PD1 targeting component, and The Fc domain contains a first Fc polypeptide and a second Fc polypeptide. The first Fc polypeptide contains amino acid substitutions of S444P, P445L, and G447L to engineer tumor-associated protease cleavage sites, while the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and The first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the attenuated IL-2 polypeptide.

21. A masking cytokine comprising: A weakened interleukin-2 (IL-2) polypeptide containing amino acid substitutions at R38A, F42A, Y45A, E62A, and C125A. The VHH masking portion includes CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16). Anti-PD1 targeting component, and The Fc domain contains a first Fc polypeptide and a second Fc polypeptide. The first Fc domain contains amino acid substitutions of S444P, P445L, and G447L to engineered tumor-associated protease cleavage sites, and the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and The first Fc domain is connected to the VHH masking portion and the second Fc domain is connected to the attenuated IL-2 peptide.

22. A masking cytokine comprising: A weakened interleukin-2 (IL-2) polypeptide containing amino acid substitutions at R38A, F42A, Y45A, E62A, and C125A. The VHH masking portion includes CDR1 of the sequence GSIFSINVMG (SEQ ID NO: 14), CDR2 of the sequence AISSGGSTNYADSVKG (SEQ ID NO: 15), and CDR3 of the sequence ASSWYEDETDY (SEQ ID NO: 16). Anti-PD1 targeting component, and The Fc domain contains a first Fc polypeptide and a second Fc polypeptide. The first Fc domain comprises amino acid substitutions of S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P with engineered tumor-associated protease cleavage sites, and the second Fc polypeptide does not contain tumor-associated protease cleavage sites; and The first Fc domain is connected to the VHH masking portion and the second Fc domain is connected to the attenuated IL-2 peptide.

23. The masked cytokine of any one of claims 1 to 22, wherein the VHH comprises the amino acid sequence AAA (SEQ ID NO: 18) at its C-terminus.

24. The masked cytokine of any one of claims 17 or 19, 21 to 22, wherein the VHH comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 19).

25. The masked cytokine of any one of claims 17 or 19, 21 to 23, wherein the VHH comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCMYASSWYEDETDYWGQGTQVTVSSAAA (SEQ ID NO: 21).

26. The masked cytokine of any one of claims 18, 20 or 23, wherein the VHH comprises the amino acid sequence of EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKGRELVAAISSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAYASSFYEDETDYWGQGTQVTVSS (SEQ ID NO: 20).

27. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises The heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 43), The heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 44), The heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 45), The light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 46), The light chain CDR2 sequence of LAS (SEQ ID NO: 47), and The light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 48).

28. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises a heavy chain variable region having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

41.

29. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises a heavy chain variable region having an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

42.

30. The masking cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises: Contains the heavy chain variable region of SEQ ID NO: 41, and It contains the light chain variable region of SEQ ID NO:

42.

31. The masking cytokine of any one of claims 1 to 18 or 23 to 30, wherein the second Fc polypeptide does not contain the tumor-associated protease cleavage site.

32. The masking cytokine of claim 31, wherein the first Fc polypeptide is linked to the IL-2 polypeptide, and the second Fc polypeptide is linked to the masking portion.

33. The masking cytokine of claim 31, wherein the first Fc polypeptide is linked to the masking portion and the second Fc polypeptide is linked to the IL-2 polypeptide.

34. The masking cytokine of any one of claims 31 to 33, wherein the first Fc polypeptide comprises amino acid substitutions of EU numbers S442G, L443G, S444P, P445L, and G447L.

35. The masking cytokine of any one of claims 31 to 33, wherein the first Fc polypeptide comprises amino acid substitutions of EU numbers S444P, P445L, and G447L.

36. The masked cytokine of any one of claims 31 to 33, wherein the first Fc polypeptide comprises amino acid substitutions of EU numbers S440M, L441P, S442Y, L443D, S444L, P445Y, G446H, and G447P.

37. The masking cytokine of any one of claims 31 to 33, wherein the first Fc polypeptide comprises amino acid substitutions of EU numbers Q438A, K439P, S440A, L441G, S442L, L443I, S444V, G446Y, and G447N.

38. The masking cytokine of any one of claims 31 to 33, wherein the first Fc polypeptide comprises amino acid substitutions of EU numbers Q438P, K439A, S440N, S442V, L443A, S444P, P445D, and G446P.

39. The masking cytokine of any one of claims 19 to 38, wherein the first Fc polypeptide and / or the second Fc polypeptide comprises an amino acid substitution of N297A.

40. The masking cytokine of any one of claims 19 to 39, wherein the first Fc polypeptide or the second Fc polypeptide comprises amino acid substitutions of H435R and Y436F.

41. The masking cytokine of any one of claims 19 to 40, wherein the first Fc polypeptide comprises amino acid substitutions of Y349C, T366S, L368A, and Y407V.

42. The masking cytokine of any one of claims 19 to 41, wherein the second Fc polypeptide comprises amino acid substitutions of S354C and T366W.

43. The masking cytokine of any one of claims 19 to 42, wherein the first Fc polypeptide comprises SEQ ID NO: 23, and the second Fc polypeptide comprises SEQ ID NO:

33.

44. The masked cytokine of any one of claims 19 to 42, wherein the first Fc polypeptide comprises SEQ ID NO: 24, and the second Fc polypeptide comprises SEQ ID NO:

33.

45. The masking cytokine of any one of claims 19 to 42, wherein the first Fc polypeptide comprises SEQ ID NO: 25, and the second Fc polypeptide comprises SEQ ID NO:

33.

46. ​​The masking cytokine of any one of claims 19 to 42, wherein the first Fc polypeptide comprises SEQ ID NO: 26, and the second Fc polypeptide comprises SEQ ID NO:

33.

47. The masking cytokine of any one of claims 19 to 42, wherein the first Fc polypeptide comprises SEQ ID NO: 27, and the second Fc polypeptide comprises SEQ ID NO:

33.

48. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide containing at least 90% of the same amino acid sequence as SEQ ID NO:

13. The VHH masked portion contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

19. The anti-PD1 targeting region includes a variable heavy chain region (VH) that is at least 90% identical to that of SEQ ID NO: 41, and a variable light chain region (VL) that is at least 90% identical to that of SEQ ID NO:

42. The first Fc polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 27, and The second Fc polypeptide contains SEQ ID NO: 33, The first Fc domain is connected to the VHH masking portion and the second Fc domain is connected to the IL-2 peptide.

49. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide containing at least 90% of the same amino acid sequence as SEQ ID NO:

13. The VHH masked portion contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

21. The anti-PD1 targeting region includes a variable heavy chain region (VH) that is at least 90% identical to that of SEQ ID NO: 41, and a variable light chain region (VL) that is at least 90% identical to that of SEQ ID NO:

42. The first Fc polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 27 or SEQ ID NO: 23, and The second Fc polypeptide contains SEQ ID NO: 33, The first Fc domain is connected to the VHH masking portion and the second Fc domain is connected to the IL-2 peptide.

50. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide containing at least 90% of the same amino acid sequence as SEQ ID NO:

12. The VHH masked portion contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

19. The anti-PD1 targeting region includes a variable heavy chain region (VH) that is at least 90% identical to that of SEQ ID NO: 41, and a variable light chain region (VL) that is at least 90% identical to that of SEQ ID NO:

42. The first Fc polypeptide comprises an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26, and The second Fc polypeptide contains SEQ ID NO: 33, The first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the IL-2 polypeptide.

51. A masking cytokine comprising: Interleukin-2 (IL-2) polypeptide containing at least 90% of the same amino acid sequence as SEQ ID NO: 11, The VHH masked portion contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

19. The anti-PD1 targeting region includes a variable heavy chain region (VH) that is at least 90% identical to that of SEQ ID NO: 41, and a variable light chain region (VL) that is at least 90% identical to that of SEQ ID NO:

42. The first Fc polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO: 23, and The second Fc polypeptide contains SEQ ID NO: 33, The first Fc polypeptide is linked to the VHH masking portion and the second Fc polypeptide is linked to the IL-2 polypeptide.

52. The masking cytokine of any one of claims 48 to 51, wherein the IL-2 polypeptide comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

11.

53. The masked cytokine of claim 52, wherein the IL-2 polypeptide comprises the same amino acid sequence as SEQ ID NO: 11100%.

54. The masking cytokine of any one of claims 48 to 51, wherein the IL-2 polypeptide comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

12.

55. The masked cytokine of claim 54, wherein the IL-2 polypeptide comprises the same amino acid sequence as SEQ ID NO: 12100%.

56. The masking cytokine of any one of claims 48 to 51, wherein the IL-2 polypeptide comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

13.

57. The masked cytokine of claim 56, wherein the IL-2 polypeptide comprises the same amino acid sequence as SEQ ID NO: 13100%.

58. The masking cytokine of any one of claims 48 to 57, wherein the VHH masking portion comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

19.

59. The masking cytokine of claim 58, wherein the VHH masking portion comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

19.

60. The masking cytokine of any one of claims 48 to 57, wherein the VHH masking portion comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

20.

61. The masking cytokine of claim 60, wherein the VHH masking portion comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

20.

62. The masking cytokine of any one of claims 48 to 57, wherein the VHH masking portion comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

21.

63. The masking cytokine of claim 62, wherein the VHH masking portion comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

21.

64. The masked cytokine of any one of claims 48 to 63, wherein the VH comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the amino acid sequence identical to SEQ ID NO: 41, and the VL comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the amino acid sequence identical to SEQ ID NO:

42.

65. The masked cytokine of claim 64, wherein the VH comprises the amino acid sequence of SEQ ID NO: 41, and the VL comprises the amino acid sequence of SEQ ID NO:

42.

66. The masking cytokine of any one of claims 48 to 65, wherein the first Fc polypeptide comprises at least 90%, 93%, 95%, 97%, 98%, or 99% of the same amino acid sequence as SEQ ID NO:

23.

67. The masked cytokine of claim 66, wherein the first Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

23.

68. The masked cytokine of any one of claims 48 to 65, wherein the first Fc domain comprises an amino acid sequence that is at least 90%, 93%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

24.

69. The masked cytokine of claim 68, wherein the first Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

24.

70. The masked cytokine of any one of claims 48 to 65, wherein the first Fc domain comprises an amino acid sequence that is at least 90%, 93%, 95%, 97%, 98%, or 99% identical to that of SEQ ID NO:

25.

71. The masked cytokine of claim 70, wherein the first Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

25.

72. The masked cytokine of any one of claims 48 to 65, wherein the first Fc domain comprises an amino acid sequence that is at least 90%, 93%, 95%, 97%, 98%, or 99% identical to that of SEQ ID NO:

26.

73. The masked cytokine of claim 72, wherein the first Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

26.

74. The masked cytokine of any one of claims 48 to 65, wherein the first Fc domain comprises an amino acid sequence that is at least 90%, 93%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:

27.

75. The masked cytokine of claim 74, wherein the first Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

27.

76. The masked cytokine of any one of claims 48 to 75, wherein the second Fc domain comprises an amino acid sequence that is at least 90%, 93%, 95%, 97%, 98%, or 99% identical to that of SEQ ID NO:

33.

77. The masked cytokine of claim 76, wherein the second Fc domain comprises an amino acid sequence that is 100% identical to that of SEQ ID NO:

33.

78. A masking cytokine comprising: The first polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

63. The second polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

74. The third polypeptide contains at least 90% of the same amino acid sequence as SEQ ID NO:

50.

79. A masking cytokine comprising: The first polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

65. The second polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

68. The third polypeptide contains at least 90% of the same amino acid sequence as SEQ ID NO:

50.

80. A masking cytokine comprising: The first polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

65. The second polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

67. The third polypeptide contains at least 90% of the same amino acid sequence as SEQ ID NO:

50.

81. A masking cytokine comprising: The first polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

64. The second polypeptide contains an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

66. The third polypeptide contains at least 90% of the same amino acid sequence as SEQ ID NO:

50.

82. The masked cytokine of claim 78, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 67, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

50.

83. The masked cytokine of claim 78, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 63, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 74, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

50.

84. The masked cytokine of claim 79, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 65, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 68, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

50.

85. The masked cytokine of claim 80, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 65, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 67, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

50.

86. The masked cytokine of claim 81, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO: 64, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 66, and the third polypeptide comprises the amino acid sequence of SEQ ID NO:

50.

87. A nucleic acid encoding a masked cytokine as described in any of the preceding claims.

88. A vector comprising the nucleic acid of claim 87.

89. A host cell comprising the nucleic acid of claim 87 or the vector of claim 88.

90. A method for producing a masked cytokine according to any one of claims 1 to 86, the method comprising culturing the host cell of claim 89 under conditions for producing the masked cytokine.

91. A pharmaceutical composition comprising any one of claims 1 to 86 a masking cytokine and a pharmaceutically acceptable carrier.

92. A kit comprising any one of claims 1 to 86 a masked cytokine or the pharmaceutical composition of claim 91.

93. A method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of any one of claims 1 to 86 of a masked cytokine or the pharmaceutical composition of claim 91.

94. A method for treating or preventing an inflammatory or autoimmune disease in a subject, the method comprising administering to the subject an effective amount of any one of claims 1 to 86 of a masked cytokine or the pharmaceutical composition of claim 91.

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