Masked il-2 cytokines and methods of use thereof

Abstract

The present invention provides, among other things, a masked cytokine comprising an attenuated interleukin 2 (IL-2) polypeptide, a VHH masking moiety, a carrier moiety, and an anti-PD1 targeting moiety. In some embodiments, the IL-2 polypeptide is engineered with a proteolytically cleavable linker comprising to be activated at a target site, such as in a tumor microenvironment, by protease activity. 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. When the cleavable linker is proteolytically cleaved at the target site, the IL-2 polypeptide is activated, which enables it to bind to its cognate receptor or protein with increased affinity.

Description

Cross-references to related applications

[0001] This application claims priority and benefit to U.S. Provisional Application No. 63 / 579,684, filed August 30, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] References to sequence lists are incorporated This specification relates to and includes the sequence list submitted therewith (submitted electronically on August 30, 2024, as an XML file named XTX_IL2_04WO1_SL.xml). This .xml file was generated on August 28, 2024, and is 171,326 bytes in size. The entire contents of the sequence list are incorporated herein by reference in their entirety. 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, particularly in targeted therapies, much work remains to be done in this field. Immunotherapy, and a branch of this field called immuno-oncology, are creating viable and exciting treatment options for malignant tumors. Specifically, it is now recognized that a hallmark of cancer is immune escape, and substantial efforts have been made to identify targets and develop therapies against these targets to reactivate the immune system to recognize and treat cancer.

[0004] Cytokine therapy is an effective strategy for stimulating the immune system to induce anti-tumor cytotoxicity. Specifically, Proleukin (aldesleukin) (a recombinant form of interleukin-2 (IL-2)) has been approved by the FDA for the treatment of metastatic renal cell carcinoma and melanoma. Unfortunately, cytokines administered to patients typically have very short half-lives, thus requiring frequent dosing. For example, the product label for aldesleukin, marketed under the brand name Proleukin, indicates that the drug shows a half-life of 85 minutes in patients receiving a 5-minute intravenous (IV) infusion. Furthermore, administration of high doses of cytokines can cause adverse health outcomes, such as vascular leakage, through systemic immune activation. These findings highlight the need to develop IL-2 peptide therapeutics that effectively target tumors without the side effects associated with systemic immune activation. Summary of the Invention

[0005] The present invention particularly provides a masked cytokine comprising an attenuated IL-2 and a VHH masking moiety. The VHH masking moiety binds to the IL-2 cytokine and inhibits the biological activity of the cytokine in an undesirable target. Upon cleavage in a desired target (e.g., a tumor), the masking moiety is released from the cytokine, activating the function of the IL-2 polypeptide. In particular, the masked cytokine of the present invention comprising an attenuated IL-2 and a VHH masking moiety is characterized by (1) efficient masking efficiency, such as inhibition of the function of the IL-2 cytokine in an undesirable target; (2) efficient activation of IL-2 by a protease to release the VHH masking moiety; (3) selective activation of IL-2 in the tumor 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 attenuated IL-2 polypeptide, a masking portion comprising a heavy-chain-only antibody (VHH), a carrier portion, and an anti-PD1 targeting portion.

[0007] In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position R38 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position F42 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position K43 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position Y45 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position E62 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 polypeptide contains a mutation at amino acid position L72 compared to the sequence of mature IL-2 having SEQ ID NO: 2. In some embodiments, the attenuated IL-2 peptide contains a mutation at amino acid position C125 compared to the sequence of mature IL-2 having SEQ ID NO: 2.

[0008] In some embodiments, the attenuated IL-2 peptide contains F42E and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains E62S and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains F42A and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains Y45R and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains F42S, E62S, and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains the F42K mutation. In some embodiments, the attenuated IL-2 peptide contains F42A, Y45A, L72G, and C125A mutations. In some embodiments, the attenuated IL-2 peptide contains the E62R mutation. In some embodiments, the attenuated IL-2 peptide contains the Y45N mutation. In some embodiments, the attenuated IL-2 peptide contains the K43A mutation. In some embodiments, the attenuated IL-2 peptide contains Y45A and E62S mutations. In some embodiments, the attenuated IL-2 peptide contains R38G, Y45A, and E62S mutations. In some embodiments, the attenuated IL-2 peptide contains a C125A mutation.

[0009] In some implementations, the attenuated IL-2 peptide contains SEQ ID NO: 4.

[0010] In some implementations, VHH specifically binds to the IL-2 peptide.

[0011] In some embodiments, VHH comprises HCDR1 containing the amino acid sequence GSIFSINVMG (SEQ ID NO: 6), HCDR2 containing the amino acid sequence AISSGGSTNYADSVKG (SEQ ID NO: 7), and HCDR3 containing an amino acid sequence selected from ASSWYEDETDY (SEQ ID NO: 8).

[0012] In some embodiments, VHH comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 5. In some embodiments, VHH comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 5.

[0013] In some implementations, VHH comprises the amino acid sequence of SEQ ID NO: 5.

[0014] In some embodiments, the VHH includes an amino acid extension. In some embodiments, the extension is located at the N-terminus of the VHH. In some embodiments, the extension is located at the C-terminus of the VHH.

[0015] In some embodiments, the amino extension comprises one amino acid. In some embodiments, the amino extension comprises two amino acids. In some embodiments, the amino extension comprises three amino acids. In some embodiments, the amino extension comprises four amino acids. In some embodiments, the amino extension comprises five amino acids. In some embodiments, the amino extension comprises six amino acids. In some embodiments, the amino extension comprises seven amino acids. In some embodiments, the amino extension comprises eight amino acids. In some embodiments, the amino extension comprises nine amino acids. In some embodiments, the amino extension comprises ten amino acids.

[0016] In some embodiments, the amino acid extension is residue A. In some embodiments, the amino acid extension is residue AS. In some embodiments, the amino acid extension is residue AST. In some embodiments, the amino acid extension is residue AAA. In some embodiments, the amino acid extension is residue AH. In some embodiments, the amino acid extension is residue GS. In some embodiments, the amino acid extension is residue PP. In some embodiments, the amino acid extension is residue PPP. In some embodiments, the amino acid extension is residue GP.

[0017] In some embodiments, VHH comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 88% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 12. In some embodiments, VHH comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 12.

[0018] In some implementations, VHH comprises the amino acid sequence of SEQ ID NO: 12.

[0019] In some embodiments, the targeting portion comprises an antigen-binding domain. In some embodiments, the targeting portion comprises a 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. In some embodiments, the targeting portion comprises a combination of antigen-binding fragments.

[0020] In some embodiments, the targeting portion comprises the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 172), the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 173), and the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 174). In some embodiments, the targeting portion comprises the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 177), the light chain CDR2 sequence of LAS (SEQ ID NO: 178), and the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 179).

[0021] In some embodiments, the targeting portion comprises the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 172), the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 173), the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 174), the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 177), the light chain CDR2 sequence of LAS (SEQ ID NO: 178), and the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 179).

[0022] In some embodiments, the targeting portion includes a heavy chain variable region having at least about 80% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 82% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 85% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 88% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 89% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 90% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 91% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 92% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 93% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 94% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 95% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 96% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 97% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 98% identity with SEQ ID NO: 169. In some embodiments, the targeting portion includes a heavy chain variable region having at least about 99% identity with SEQ ID NO: 169.

[0023] In some embodiments, the targeting portion includes a light chain variable region having at least about 80% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 82% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 85% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 88% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 89% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 90% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 91% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 92% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 93% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 94% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 95% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 96% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 97% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 98% identity with SEQ ID NO: 175. In some embodiments, the targeting portion includes a light chain variable region having at least about 99% identity with SEQ ID NO: 175.

[0024] In some embodiments, the targeting portion includes a heavy chain region having at least about 80% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 82% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 85% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 88% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 89% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 90% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 91% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 92% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 93% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 94% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 95% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 96% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 97% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 98% identity with SEQ ID NO: 170. In some embodiments, the targeting portion includes a heavy chain region having at least about 99% identity with SEQ ID NO: 170.

[0025] In some embodiments, the targeting portion includes a light chain region having at least about 80% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 82% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 85% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 88% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 89% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 90% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 91% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 92% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 93% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 94% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 95% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 96% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 97% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 98% identity with SEQ ID NO: 176. In some embodiments, the targeting portion includes a light chain region having at least about 99% identity with SEQ ID NO: 176.

[0026] In some embodiments, the targeting portion includes the heavy chain variable region of SEQ ID NO: 169 and the light chain variable region of SEQ ID NO: 175. In some embodiments, the targeting portion includes the heavy chain region of SEQ ID NO: 170 and the light chain region of SEQ ID NO: 176.

[0027] In some embodiments, the vector portion comprises a PEG molecule. In some embodiments, the vector portion comprises albumin. In some embodiments, the vector portion comprises an albumin fragment. In some embodiments, the vector portion comprises an antibody Fc domain. In some embodiments, the vector portion comprises an antibody. In some embodiments, the vector portion comprises an antigen-binding fragment.

[0028] In some implementations, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide.

[0029] In some embodiments, a first Fc polypeptide is linked to an IL-2 polypeptide via a first connector, and a second Fc polypeptide is linked to a masking portion via a second connector, wherein the first or second connector contains a tumor-associated protease cleavage site, causing the masking portion to be released from the IL-2 polypeptide.

[0030] In some embodiments, a first Fc polypeptide is linked to a masking portion via a first connector, and a second Fc polypeptide is linked to an IL-2 polypeptide via a second connector, wherein the first or second connector contains a tumor-associated protease cleavage site, causing the masking portion to be released from the IL-2 polypeptide.

[0031] In some embodiments, the first connective includes a tumor-associated protease cleavage site. In some embodiments, the second connective includes a tumor-associated protease cleavage site. In some embodiments, the first Fc polypeptide includes Y349C, T366S, L368A, and Y407V mutations, and the second Fc polypeptide includes S354C and T366W mutations. In some embodiments, the second Fc polypeptide includes Y349C, T366S, L368A, and Y407V mutations, and the first Fc polypeptide includes S354C and T366W mutations.

[0032] In some embodiments, the first Fc polypeptide includes a modified CH3 domain that weakens or eliminates binding to protein A, and the second Fc polypeptide includes a CH3 domain that binds to protein A.

[0033] In some implementations, modifications that weaken or eliminate binding to protein A, based on the Kabat number, are H435R and Y436F.

[0034] In some embodiments, the first Fc polypeptide contains the N297A mutation. In some embodiments, the second Fc polypeptide contains the N297A mutation. In some embodiments, both the first and second Fc polypeptides contain the N297A mutation.

[0035] In some embodiments, the first Fc polypeptide comprises an IgG1 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises an IgG2 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide comprises an IgG4 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises an IgG1 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises an IgG2 Fc domain or a fragment thereof. In some embodiments, the second Fc polypeptide comprises an IgG4 Fc domain or a fragment thereof.

[0036] In some embodiments, the first Fc polypeptide contains an I253A mutation. In some embodiments, the second Fc polypeptide contains an I253A mutation. In some embodiments, both the first and second Fc polypeptides contain an I253A mutation.

[0037] In some embodiments, the first Fc polypeptide contains the S354C, T366W, N297A, H435R, and Y435F mutations, and the second Fc polypeptide contains the Y349C, T366S, L368A, Y407V, and N297A mutations.

[0038] In some embodiments, the tumor-associated protease cleavage site comprises a sequence selected from Table 3. The tumor-associated protease cleavage site comprises MPYDLYHP (SEQ ID NO: 34).

[0039] In one aspect, the present invention particularly provides a masked cytokine comprising (a) an attenuated interleukin-2 (IL-2) polypeptide comprising amino acid substitutions of F42E and C125A; (b) a VHH masking portion comprising CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO: 8; (c) an Fc domain comprising a first Fc polypeptide linked to the attenuated IL-2 polypeptide via a first linker and a second Fc polypeptide linked to the VHH masking portion via a second linker; and (d) an anti-PD1 targeting portion.

[0040] In one aspect, the present invention particularly provides a masked cytokine comprising (a) an attenuated interleukin-2 (IL-2) polypeptide comprising SEQ ID NO: 4; (b) a VHH masking portion comprising SEQ ID NO: 5 or SEQ ID NO: 12; (c) an Fc domain comprising a first Fc polypeptide of SEQ ID NO: 21 linked to the attenuated IL-2 polypeptide via a first linker and a second Fc polypeptide of SEQ ID NO: 20 linked to the VHH masking portion via a second linker; and (d) an anti-PD1 targeting domain portion comprising SEQ ID: 169 and 175.

[0041] In one aspect, the present invention particularly provides a masked cytokine comprising three polypeptides: (a) a first polypeptide comprising a variable heavy chain of an anti-PD1 targeting moiety, a first Fc polypeptide, an uncleavable linker, and an attenuated IL-2 polypeptide; (b) a second polypeptide comprising a variable heavy chain of an anti-PD1 targeting moiety, a second Fc polypeptide, a cleavable linker, and a VHH masking moiety; and (c) a third polypeptide comprising a variable light chain of an anti-PD1 targeting moiety.

[0042] In some embodiments, the first Fc polypeptide comprises SEQ ID NO: 21, and the second Fc polypeptide comprises SEQ ID NO: 20.

[0043] In some embodiments, the variable heavy chain of the anti-PD-1 targeting portion comprises hCDR1 of SEQ ID NO: 172, hCDR2 of SEQ ID NO: 173, and hCDR3 of SEQ ID NO: 174. In some embodiments, the variable light chain of the anti-PD-1 targeting portion comprises lCDR1 of SEQ ID NO: 177, lCDR2 of SEQ ID NO: 178, and lCDR3 of SEQ ID NO: 178. In some embodiments, the variable heavy chain of the anti-PD-1 targeting portion comprises hCDR1 of SEQ ID NO: 172, hCDR2 of SEQ ID NO: 173, and hCDR3 of SEQ ID NO: 174, and the variable light chain of the anti-PD-1 targeting portion comprises lCDR1 of SEQ ID NO: 177, lCDR2 of SEQ ID NO: 178, and lCDR3 of SEQ ID NO: 179.

[0044] In some embodiments, the variable heavy chain of the anti-PD-1 targeting portion comprises SEQ ID NO: 169. In some embodiments, the variable light chain of the anti-PD-1 targeting portion comprises SEQ ID NO: 175. In some embodiments, the variable heavy chain of the anti-PD-1 targeting portion comprises SEQ ID NO: 169, and the variable light chain of the anti-PD-1 targeting portion comprises SEQ ID NO: 175.

[0045] In some embodiments, the cuttable connector comprises MPYDLYHP (SEQ ID NO: 34). In some embodiments, the cuttable connector comprises SPGGGGPMPYDLYHPSGGG (SEQ ID NO: 144).

[0046] In some implementations, the non-cuttable connector comprises GGSSPPGGGSSGGGSGP (SEQ ID NO: 163).

[0047] In one aspect, the present invention particularly provides a masked cytokine comprising three polypeptides: (a) a first polypeptide comprising (i) a variable heavy chain comprising an anti-PD1 targeting moiety of SEQ ID NO: 169, (ii) a first Fc polypeptide of SEQ ID NO: 21, (iii) an uncleavable linker, and (iv) an attenuated IL-2 polypeptide of SEQ ID NO: 4, (b) a second polypeptide comprising (i) a variable heavy chain comprising an anti-PD1 targeting moiety of SEQ ID NO: 169, (ii) a second Fc polypeptide of SEQ ID NO: 20, (iii) a cleavable linker comprising MPYDLYHP (SEQ ID NO: 34), and (iv) a VHH masking moiety of SEQ ID NO: 5 or SEQ ID NO: 12, and (c) a third polypeptide comprising a variable light chain containing an anti-PD1 targeting moiety of SEQ ID NO: 175.

[0048] In some embodiments, the first polypeptide comprises SEQ ID NO: 191. In some embodiments, the second polypeptide comprises SEQ ID NO: 192. In some embodiments, the third polypeptide comprises SEQ ID NO: 176. In some embodiments, the first polypeptide comprises SEQ ID NO: 191, the second polypeptide comprises SEQ ID NO: 192, and the third polypeptide comprises SEQ ID NO: 176.

[0049] In one aspect, the present invention particularly provides a masked cytokine comprising three polypeptides: (a) a first polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 191; (b) a second polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 193; and (c) a third polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 176.

[0050] In some embodiments, the first polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 191.

[0051] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 193.

[0052] In some embodiments, the third polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 176.

[0053] In one aspect, the present invention particularly provides a masked cytokine comprising three polypeptides: (a) a first polypeptide comprising the amino sequence of SEQ ID NO: 191, (b) a second polypeptide comprising the amino sequence of SEQ ID NO: 193, and (c) a third polypeptide comprising the amino sequence of SEQ ID NO: 176.

[0054] In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 50 times greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 100 times greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 120 times greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 150 times greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 200 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 250 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 280 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 300 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 350 times greater than that of the unmasked cytokine. In some embodiments, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is at least 400 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 450 times greater than that of the unmasked cytokine. In some embodiments, the EC50 of the masked cytokine is at least 500 times greater than that of the unmasked cytokine.

[0055] In some implementations, the masked cytokines have a concentration greater than or equal to 10 in the presence of MMPs. 4 M -1 s -1 The in vitro cleavage efficiency Kcat / Km. In some embodiments, the masked cytokines have a cleavage efficiency greater than or equal to 5 x 10 in the presence of MMPs. 4 M -1 s-1 The in vitro cleavage efficiency Kcat / Km. In some embodiments, the masked cytokines have a concentration greater than or equal to 10 in the presence of MMPs. 5 M -1 s -1 The in vitro cleavage efficiency Kcat / Km. In some embodiments, the masked cytokines have a cleavage efficiency greater than or equal to 5 x 10 in the presence of MMPs. 5 M -1 s -1 The in vitro cleavage efficiency Kcat / Km. In some embodiments, the masked cytokines have a concentration greater than or equal to 10 in the presence of MMPs. 6 M -1 s -1 The external cutting efficiency Kcat / Km.

[0056] In some embodiments, at least 3% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 5% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 10% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 15% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 20% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 25% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 30% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 35% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 40% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 45% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 50% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 55% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 60% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 65% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 70% of the masked cytokines are cleaved upon contact with tumor-specific proteases. In some embodiments, at least 75% of the masked cytokines are cleaved upon contact with tumor-specific proteases.

[0057] In one aspect, the present invention particularly provides a nucleic acid that encodes the masked cytokines described herein.

[0058] In one aspect, the present invention provides a carrier comprising nucleic acid encoding the masking cytokine described herein.

[0059] In one aspect, the present invention particularly provides a host cell comprising the vector described herein.

[0060] In one aspect, the present invention particularly provides a method for producing the masked cytokines described herein, wherein host cells are cultured under conditions that produce the masked cytokines.

[0061] In one aspect, the present invention particularly provides a pharmaceutical composition comprising the masked cytokines described herein and a pharmaceutically acceptable carrier.

[0062] In one aspect, the present invention particularly provides a kit containing the masked cytokines described herein.

[0063] 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 masked cytokine or pharmaceutical composition described herein.

[0064] 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 masked cytokine or pharmaceutical composition described herein.

[0065] In one aspect, the present invention provides a masked cytokine as described herein, which is used in medicine.

[0066] In one aspect, the present invention provides a masked cytokine as described herein for use in the treatment of neoplastic diseases.

[0067] In one aspect, the present invention provides a masked cytokine as described herein for the treatment or prevention of inflammatory diseases.

[0068] In one aspect, the present invention provides a masked cytokine as described herein for use in the treatment or prevention of autoimmune diseases. Attached Figure Description

[0069] The accompanying drawings are for illustrative purposes only and are not intended to be limiting.

[0070] Figures 1A-1B A schematic diagram of an exemplary masked IL-2 cytokine construct is shown. Figure 1AIn the first polypeptide (chain A), there is a targeting moiety, an Fc domain with "groove" and RF mutations, and an IL-2 polypeptide. The second polypeptide (chain B) contains a targeting moiety, an Fc domain with a "groove" mutation, a cleavable linker, and an anti-IL-2 VHH as a masking moiety. Figure 1A The masked cytokines in the formula contain a third and a fourth polypeptide (two copies of chain C), which comprises a variable light chain (VL) and a constant light chain (CL). Figure 1B In the first polypeptide (chain A), there is a targeting moiety, an Fc domain with "groove" and RF mutations, an IL-2 polypeptide, and a cleavable linker. The second polypeptide (chain B) contains a targeting moiety, an Fc domain with a "groove" mutation, and an anti-IL-2 VHH as a masking moiety. Figure 1A The masked cytokine contains a third and a fourth polypeptide (two copies of chain C), which comprises a variable light chain (VL) and a constant light chain (CL). The cross "X" indicates the location of the cleavage site.

[0071] Figure 2 An exemplary in vitro measurement of pSTAT5 activity in hPBMC in response to targeted masked cytokine activity is shown.

[0072] Figure 3A This is an exemplary graph showing the percentage of molecules cleaved by targeted, masked cytokines in tumor cells of different cancer indications. The cancer indications are plotted in the following order: melanoma, HNC (head and neck cancer), lung cancer, colon cancer, and RCC (renal cell carcinoma). Figure 3B This is an exemplary graph showing the percentage of molecules cleaved by targeted, masked cytokines in plasma from different cancer indications.

[0073] Figure 4 Exemplary measurements of CD8 T cell profiles in response to targeted masked cytokines are shown in spleen cells and tumor microenvironments, respectively.

[0074] Figure 5 An exemplary graph illustrating the percentage of in vivo active molecules produced by protease cleavage of VHH-masked portions in tumors, plasma, and spleen is shown.

[0075] Figure 6 This is an exemplary graph showing the tumor volume of mice inoculated with tumors during a 15-day treatment period using the C5 and C6 constructs. The graph shows the tumor growth inhibition percentage for each treatment group.

[0076] Figure 7A This is an exemplary diagram showing the tumor volume of mice inoculated with tumors during a 15-day treatment period. Figure 7B These are a series of exemplary graphs showing survival statistics for mice given various treatments.

[0077] Figure 8 This is an exemplary graph illustrating the fold induction of TCR signaling by an exemplary masked IL-2 cytokine in a PD-1 / PD-L1 blockade bioassay.

[0078] definition

[0079] To facilitate understanding of the invention, certain terms are defined below. Further definitions of the following and other terms are set forth throughout this specification. Publications and other references cited herein to describe the background of the invention and to provide further details regarding its implementation are incorporated herein by reference.

[0080] It should be understood that the present invention is not limited to specific compositions or biological systems, although they may be modified. 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.

[0081] Unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in this specification and the appended claims include plural indicators. Thus, for example, reference to “an IL-2 polypeptide” optionally includes a combination of two or more such polypeptides, and so on.

[0082] 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 for that value or parameter itself.

[0083] It should be understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “comprising”, “consisting”, and “consisting essentially of”.

[0084] 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 implementations: 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).

[0085] The term "antibody" includes polyclonal antibodies, monoclonal antibodies (including full-length antibodies having the Fc region of an immunoglobulin), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, 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.

[0086] 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 those antibodies that have both a heavy chain and a light chain, including 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, whole antibodies may possess one or more effector functions.

[0087] "Antibody fragment" comprises a portion of a complete antibody, such as the antigen-binding region and / or variable region of the complete antibody, and / or the constant region of the complete antibody. Examples of antibody fragments include the Fc region of an antibody, a portion of the 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; bivalent 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, namely single-chain Fv (scFv), single-domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single-domain antibody-linked Fab (s-Fab), 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.

[0088] 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 vacancies (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 (e.g., 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 algorithm 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:

[0089] 100 multiplied by the fraction X / Y

[0090] Where X is the number of amino acid residues that are considered identical matches in the sequence alignments of A and B by the program, and Y is the total number of amino acid residues in B. It should be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity percentage of A to B will not be equal to the amino acid sequence identity percentage of B to A.

[0091] Antibody "effective function" refers to those biological activities attributed to the antibody's Fc region (either the native Fc region or the Fc region with amino acid sequence variations), and these activities 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.

[0092] "Attenuated IL-2" is used herein to refer to an IL-2 variant containing one or more mutations that weaken, but do not eliminate, binding to IL-2Rα. The inventors have found that "attenuated IL-2" more effectively expands tumor-specific T cells compared to "non-α IL-2" and exhibits lower regulatory T cell activity compared to its "α-biased IL-2" counterpart. In some embodiments, the attenuated IL-2 contains mutations in F42E and C125A.

[0093] “Non-α IL-2” is used herein to refer to an IL-2 variant that contains one or more mutations that eliminate the binding of IL-2Rα. In some embodiments, non-α IL-2 contains mutations of R38A, F42A, Y45A, E62A, and C125A relative to wild-type IL-2.

[0094] “α-biased IL-2” is used in this document to refer to an IL-2 variant that contains one or more mutations that weaken 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).

[0095] 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.

[0096] The “isolated” nucleic acid molecule encoding the cytokine peptides described herein is a nucleic acid molecule identified and isolated from at least one contaminant nucleic acid molecule typically associated with its environment of origin. In some embodiments, the isolated nucleic acid does not associate with all components associated with the environment of origin. The isolated nucleic acid molecule encoding the peptides and cytokine peptides described herein is in a form different from its form or environment found in nature. Therefore, the isolated nucleic acid molecule is distinguished from the nucleic acids naturally occurring in cells that encode the peptides and cytokine peptides described herein.

[0097] The term "pharmaceutical formulation" refers to a formulation in a form that enables the bioactivity of the active ingredient to be effective and that does not contain any additional components that would have unacceptable toxicity to the subject to whom the formulation will be administered.

[0098] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cells in a clinicopathological process. Ideal treatment outcomes include slowing 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 reduced or eliminated. Similarly, an individual is successfully "treated" if treatment improves their quality of life, reduces the dosage of other medications needed to treat the disease, decreases the frequency of disease relapses, alleviates the severity of the disease, slows the development or progression of the disease, and / or prolongs the individual's survival.

[0099] As used in this article, "in conjunction with" or "in combination with" means applying a treatment modality in addition to another treatment modality. Therefore, "in conjunction with" or "in combination with" means applying a treatment modality before, during, or after applying another treatment modality to an individual.

[0100] As used herein, the term "prevention" includes providing prevention against the onset or recurrence of a disease in an individual. An individual may be susceptible to, prone to, or at risk of developing a disease, but has not yet been diagnosed with it. In some implementations, the targeted cytokines described herein are used to delay disease progression.

[0101] As used herein, an individual “at risk of developing the disease” may or may not have a detectable disease or disease symptoms, and may or may not exhibit a detectable disease or disease symptoms prior to the treatments described herein. “At risk of…” indicates that an individual possesses one or more risk factors that are measurable parameters associated with the development of a disease as known in the art. An individual with one or more of these risk factors is more likely to develop the disease than an individual without one or more of these risk factors.

[0102] "Effective dose" refers to the amount that is effective in achieving the desired or specified effect (including therapeutic or preventative effects) at least at the necessary dose and for at least the necessary time period.

[0103] An effective dose can 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 disease. The therapeutic effective dose described herein may vary depending on factors such as the patient’s disease state, age, sex, weight, and the ability of the antibody to elicit the desired response in an individual. A therapeutic effective dose may also be the amount at which the therapeutically beneficial effect of the targeted cytokine outweighs any toxic or harmful effects. A “prophylactic effective dose” refers to the amount at the necessary dose and for the necessary time period to effectively achieve the desired preventative effect. Typically, but not necessarily, the prophylactic effective dose may be less than the therapeutic effective dose because the prophylactic dose is used in subjects before or in the early stages of disease.

[0104] "Chronic" administration refers to the continuous rather than acute administration of a drug to maintain the initial therapeutic effect (activity) over a prolonged period of time. "Intermittent" administration is treatment that is not performed continuously but periodically.

[0105] 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.

[0106] 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, an amino acid has the general structure H₂N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is a l-amino acid. "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 a standard amino acid, whether it is synthetically prepared or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and / or substitutes. Amino acids (including carboxyl-terminal 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 disulfide bonds. Amino acids may contain one or more post-translational modifications, such as association with one or more chemical entities (e.g., methyl, acetate, acetyl, phosphate, formyl, isoprenoid, sulfate, polyethylene glycol, lipid, carbohydrate, biotin, 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 whether it refers to free amino acids or peptide residues.

[0107] 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 clone.

[0108] Biologically active: As used herein, the phrase “biologically active” refers to the characteristic of any agent that is active in a biological system, and particularly in an organism. For example, an agent that has a biological effect on an organism when administered to that organism is considered biologically active.

[0109] Disease: As used herein, the term "disease" is a pathological condition, such as a pathological condition that can be identified by symptoms or other identifying factors as distinct from a healthy or normal state. The term "disease" includes symptoms, syndromes, symptom, and lesions. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0110] Delivery: As used herein, the term “delivery” encompasses both local and systemic delivery. For example, mRNA delivery encompasses the delivery of mRNA to a target tissue and the expression of the encoded protein, which is then retained within the target tissue (also known as “local distribution” or “local delivery”), as well as the delivery of mRNA to a target tissue and the expression of the encoded protein, which is then secreted into the patient’s circulatory system (e.g., serum) and systemically distributed and absorbed by other tissues (also known as “systemic distribution” or “systemic delivery”).

[0111] 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 disease-related symptoms are alleviated; or at least one or more disease-related biomarkers are reduced during the duration of the dosing interval. In this invention, dosing frequency and dosing interval are used interchangeably.

[0112] 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 the post-translational modification of a polypeptide or a fully assembled protein (e.g., an enzyme). In this application, the terms “expression” and “production”, as well as their grammatical equivalents, are used interchangeably.

[0113] Effective dose: As used herein, an effective dose is the dose of mRNA in a pharmaceutical composition that, when administered to a subject in need (i.e., a mammalian subject) according to the method of the invention, effectively produces the desired outcome in the subject, such as alleviating disease-related symptoms.

[0114] Host cell: As used herein, the term "host cell" is a single cell or cell culture that may be, or has been, an acceptor of any recombinant vector or isolated polynucleotide (such as a vector or polynucleotide for engineered cleavable Fc domains, or a masked cytokine of this disclosure). Host cells include cells transfected or infected in vivo or in vitro with the recombinant vectors or polynucleotides of the present invention. In some cases, the host cell is a mammalian host cell.

[0115] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, indicate a value relative to a baseline measurement, such as a measurement in the same individual prior to the initiation of the treatment described herein, or a measurement in a control subject (or multiple control subjects) in the absence of the treatment described herein. A “control subject” is a subject with the same form of disease as the subject being treated and whose age is approximately the same as that of the subject being treated.

[0116] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment (e.g., in test tubes or reaction vessels, in cell cultures, etc.) rather than in multicellular organisms.

[0117] 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 case of cell-based systems, the term may be used to refer to events that occur within living cells (rather than, for example, in vitro systems).

[0118] Part: As used in this article, the term “part” refers to a substructure that is part of a molecule.

[0119] Patient: As used herein, the term "patient" or "subject" means 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. Human includes both prenatal and postnatal forms.

[0120] Pharmaceutically acceptable: As used herein, “pharmaceutically acceptable” means a substance that, to a reasonable extent of medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0121] 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 those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid; or amino salts formed with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid; or amino salts 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, disglucuronate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucohepanoate, glycerophosphate, glucuronate, hemisulfate, heptaate, hydroiodide, 2-hydroxy-ethanesulfonate, lacturonate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthalate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, neopentanoate, 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, other 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. Other pharmaceutically acceptable salts include those formed by quaternizing amines with suitable electrophilic agents (e.g., haloalkanes) to form quaternized alkylated amino salts.

[0122] Polypeptide: As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues, such as a polymer of at least 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. Throughout this disclosure, the standard three-letter or single-letter names of amino acids are used. As used herein, the term covers amino acid chains of any length (including full-length proteins) where amino acid residues are linked by covalent peptide bonds.

[0123] Polynucleotide: As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides having a length of at least 5, 6, 7, 8, 9, or 10 bases or base pairs, is a modified form of ribonucleotides or deoxynucleotides or any type of nucleotide, and means including single-stranded and double-stranded forms of DNA and / or RNA. Throughout this disclosure, the term may be used interchangeably with "nucleic acid molecule".

[0124] 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). Human includes both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject can be a patient, which refers to a person presented to 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 or be susceptible to a disease or condition but may or may not exhibit symptoms of the disease or condition.

[0125] Essentially: As used herein, the term “essentially” refers to a qualitative condition that exhibits all or nearly all the extent / degree of the feature or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if at all) complete and / or proceed to completeness or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to indicate the possibility of lacking the completeness inherent in many biological and chemical phenomena.

[0126] 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 that exhibit disease-related pathology, symptoms, or characteristics.

[0127] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" means an amount sufficient to treat, diagnose, prevent, and / or delay the onset of symptoms of a disease, condition, and / or symptom when administered to a subject who has or is susceptible to the disease, condition, and / or symptom. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0128] Treatment: As used herein, the terms “treat,” “treatment,” or “treating” mean any method used to partially or completely reduce, improve, alleviate, suppress, prevent, delay the onset of, reduce the severity of, and / or decrease the incidence of one or more symptoms or features of a particular disease, condition, and / or symptom. Treatment may be administered to subjects who do not exhibit signs of the disease and / or only exhibit early signs of the disease for the purpose of reducing the risk of developing disease-related pathology.

[0129] Vector: As used herein, the term “vector” refers to a macromolecule or molecular complex containing polynucleotides to be delivered to a host cell or organism (in vitro or in vivo), typically a virus or plasmid.

[0130] Various aspects of the invention will be described in detail in the following sections. The use of sections is not intended to limit the invention. Each section can be applied to any aspect of the invention. In this application, unless otherwise stated, the use of "or" means "and / or". Detailed Implementation

[0131] The masked cytokine constructs described herein are engineered to optimize the binding between the masking moiety (e.g., anti-IL-2VHH) and the cytokine (e.g., IL-2 peptide), thereby promoting safe and effective targeted therapies for cancer treatment. In particular, this invention provides a masked cytokine comprising an attenuated IL-2 and a VHH masking moiety. The VHH masking moiety binds to the IL-2 cytokine and inhibits the bioactivity of the cytokine in an undesirable target. Upon cleavage in a desired target (e.g., a 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 an attenuated IL-2 and a VHH masking moiety is characterized by (1) efficient masking efficiency, such as inhibition of IL-2 cytokine function in an undesirable target; (2) efficient activation of IL-2 via a protease to release the VHH masking moiety; (3) selective activation of IL-2 in the tumor rather than in plasma; and (4) in vivo efficacy (e.g., high tumor growth inhibition).

[0132] Interleukin-2 (IL-2)

[0133] This document provides for use with any masked cytokine or its cleavage product as an IL-2 polypeptide or a functional fragment thereof. IL-2 plays a vital role in cell signaling, particularly in cells of the immune system by regulating leukocyte activity. Suitable IL-2 polypeptides used 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 substituted (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.

[0134] In eukaryotic cells, the naturally occurring IL-2 polypeptide was synthesized into a precursor polypeptide with 153 amino acids, which has SEQ ID NO: 1.

[0135] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 1)

[0136] The precursor peptide is then processed into mature IL-2 by removing amino acid residues 1-20. This results in a mature form of IL-2 consisting of 133 amino acids (amino acid residues 21-153) as described in SEQ ID NO: 2. In some embodiments, the IL-2 peptide is naturally occurring IL-2.

[0137] In some embodiments, the mature IL-2 peptide contains a mutation at position 125 relative to SEQ ID NO: 2. In some embodiments, the mutation contains C125A, resulting in SEQ ID NO: 3.

[0138] Attenuated IL-2

[0139] The inventors have discovered that the use of attenuated IL-2 is more effective at expanding tumor-specific T cells compared to other IL-2 variants (e.g., "non-α IL-2"), and exhibits lower regulatory T cell activity compared to other IL-2 variants (e.g., "α-biased IL-2"). "Attenuated IL-2" refers to an IL-2 variant containing one or more mutations that attenuate but do not eliminate binding to IL-2Rα compared to wild-type IL-2. "Non-α IL-2" refers to an IL-2 variant containing one or more mutations that eliminate binding to IL-2Rα, and "α-biased IL-2" refers to an IL-2 variant containing one or more mutations that attenuate or eliminate binding to IL-2Rβγ. In some embodiments, the attenuated IL-2 contains the F42E mutation. In some embodiments, the attenuated IL-2 contains both the F42E and C125A mutations.

[0140] 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).

[0141] In some embodiments, the IL-2 peptide or functional fragment thereof comprises an amino acid sequence having one or more amino acid substitutions that reduce the affinity of the IL-2 peptide or functional fragment thereof for IL-2Rα (CD25) compared to the amino acid sequence of wild-type IL-2 of SEQ ID NO: 2.

[0142] In some embodiments, the IL-2 polypeptide or a functional fragment thereof contains an amino acid sequence substituted for C125A, compared to the amino acid sequence of SEQ ID NO: 2.

[0143] 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. 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: 2. In some embodiments, the IL-2 polypeptide or a functional fragment thereof comprises the amino acid sequence of SEQ ID NO: 2.

[0144] In some embodiments, the IL-2 polypeptide comprises a substitution at position 42 relative to SEQ ID NO: 2. 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.

[0145] 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: 2. In some embodiments, the IL-2 peptide comprises SEQ ID NO: 3. In some embodiments, the IL-2 peptide comprises SEQ ID NO: 4.

[0146] Table 1. Exemplary IL-2 peptide sequences.

[0147]

[0148] 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., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the full-length cytokine protein). Cytokine receptor binding capacity can be demonstrated, for example, by 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).

[0149] 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 case of IL-2 peptide binding, the target protein may be IL-2R (comprising IL-2Rα, IL-2Rβ, and IL-2Rγ chains), the IL-2Rα chain, the IL-2Rβ chain, or an IL-2Rα / β dimer complex.

[0150] VHH masking section

[0151] This invention particularly provides novel anti-IL-2 antigen-binding fragments (e.g., VHH) that can be used as a masking portion of any of the masking or targeted IL-2 cytokines described herein. The VHH masking portion binds to the IL-2 cytokine and inhibits the biological activity of the cytokine. Upon cleavage, the masking portion is released from the cytokine, activating the function of the IL-2 polypeptide. 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 the non-target environment), and activation of IL-2 activity upon 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 such that the EC50 value of the masked IL-2 cytokine based on pSTAT5 activity on CD8+ T cells is greater than 300-fold compared to IL-2 cytokines without the masking portion. In some implementations, the masked cytokine exhibits an EC50 value greater than 300-fold based on pSTAT5 activity on CD8+ T cells compared to the IL-2 cytokine without the masking portion. However, IL-2 function is restored once the masking portion is cleaved from the IL-2 cytokine molecule. Preferably, the percentage of cleaved molecules in the tumor is greater than 5% in at least one tested cancer indication, and the percentage of cleaved molecules is below the lower limit of quantification (LLOQ) in plasma.

[0152] In some embodiments, the anti-IL-2 VHH of the present invention has improved stability, developability, and manufacturability for large-scale production for use in therapeutic agents. In some embodiments, the anti-IL-2 VHH of the present invention has reduced immunogenicity (i.e., minimal induction of endotoxins or reduced binding to pre-existing antidrug antibodies).

[0153] In some embodiments, anti-IL-2 VHH binds to wild-type IL-2 and non-αIL-2 with high affinity (e.g., ≤ 5 nM). In some embodiments, anti-IL-2 VHH has a lower binding affinity to α-biased IL-2 (i.e., ≥ 500 nM) relative to its binding affinity to wild-type IL-2 or non-α IL-2. In some embodiments, anti-IL-2 VHH binds to non-α IL-2 with a KD of 1 pM to 50 nM.

[0154] 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.

[0155] 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 lacking light chains. These heavy-chain antibodies contain a single variable domain (V... H VHH comprises a single-chain polypeptide with three CDRs and four frame regions (FR1-4). As used herein, a "frame region" or "FR" refers to a region within the variable domain located between the 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.

[0156] In some implementations, the VHH comprises three CDRs and four frame regions, designated as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In some implementations, the VHH may be truncated at the N-terminus or C-terminus so that it contains only a portion of FR1 and / or FR4, or lack one or both of these frame regions, as long as the VHH substantially maintains cytokine binding and specificity.

[0157] In some embodiments, the VHH masking portion is connected to the Fc domain via a cleavable connector. In some embodiments, the VHH masking portion is connected to the Fc domain via a cleavable connector as described herein (e.g., a connector comprising a cleavable peptide as described in Table 3). In some embodiments, the VHH masking portion is connected to the Fc domain via an uncleavable connector. In some embodiments, the VHH masking portion is connected to the Fc domain via an uncleavable connector as described herein.

[0158] 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.

[0159] In some implementations, the VHH antibody comprises the amino acid sequence of SEQ ID NO: 5.

[0160] EVQLVESGGGLVQPGGSLRLSCAAS GSIFSINVMG WYRQAPGKQRELVA AISSGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCMY ASSWYEDETDYWGQGTQVTVSS (SEQ ID NO: 5) – CDR with underline.

[0161] In some embodiments, the VHH antibody comprises an amino acid sequence having at least 85% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 87% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 93% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 94% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 5. In some embodiments, the VHH antibody comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 5.

[0162] In some implementations, the VHH antibody comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0163] In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by one amino acid. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by two amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by three amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by four amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by five amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by six amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by seven amino acids. In some embodiments, the VHH antibody comprises CDR1 having an amino acid sequence that differs from SEQ ID NO: 6 by eight amino acids.

[0164] In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by one amino acid. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by two amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by three amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by four amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by five amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by six amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by seven amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by eight amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by 9 amino acids. In some embodiments, the VHH antibody comprises CDR2 having an amino acid sequence that differs from SEQ ID NO: 7 by 10 amino acids.

[0165] In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by one amino acid. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by eight amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by three amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by four amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by five amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by six amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by seven amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by eight amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by 9 amino acids. In some embodiments, the VHH antibody comprises CDR3 having an amino acid sequence that differs from SEQ ID NO: 8 by 10 amino acids.

[0166] VHH amino acid elongation

[0167] In some embodiments, the VHH masking portion of the present invention is further modified. Some modifications reduce the ability of the modified molecule 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. Modifications may be selected from, for example, but not limited to, C-terminal addition, extension, deletion, or tagging. Further modifications are described in WO2013024059A2, the contents of which are incorporated herein by reference.

[0168] Furthermore, modified molecules may have an enhanced safety profile and fewer side effects compared to unmodified molecules that do not contain C-terminal extensions, additions, deletions, or tags. Administration of pharmaceutical compositions containing modified molecules can lead to 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.

[0169] In some embodiments, the targeted masking cytokine contains modifications. In some embodiments, the targeted masking cytokine contains C-terminal extension. In some embodiments, the targeted masking cytokine contains C-terminal addition. In some embodiments, the targeted masking cytokine contains C-terminal deletion. In some embodiments, the targeted masking cytokine contains a C-terminal tag.

[0170] In some embodiments, the masking portion includes embellishments. In some embodiments, the masking portion includes C-terminal extensions. In some embodiments, the masking portion includes C-terminal additions. In some embodiments, the masking portion includes C-terminal deletions. In some embodiments, the masking portion includes C-terminal tags.

[0171] 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.

[0172] In some embodiments, the C-terminal extension is selected from Table 2. In some embodiments, the C-terminal extension comprises alanine. In some embodiments, the C-terminal extension comprises the sequence AS. In some embodiments, the C-terminal extension comprises the sequence AST. In some embodiments, the C-terminal extension comprises the sequence AAA. In some embodiments, the C-terminal extension comprises the sequence AH. In some embodiments, the C-terminal extension comprises the sequence GS. In some embodiments, the C-terminal extension comprises the sequence PP. In some embodiments, the C-terminal extension comprises the sequence PPP. In some embodiments, the C-terminal extension comprises the sequence GP. In some embodiments, the C-terminal modification includes deletion and extension comprising the sequence GP.

[0173] In some embodiments, the C-terminal extension comprises alanine. In some embodiments, the C-terminal extension comprises serine. In some embodiments, the C-terminal extension comprises threonine. In some embodiments, the C-terminal extension comprises histidine. In some embodiments, the C-terminal extension comprises glycine. In some embodiments, the C-terminal extension comprises proline.

[0174] In some embodiments, the C-terminal extension comprises multiple alanine residues. In some embodiments, the C-terminal extension comprises multiple serine residues. In some embodiments, the C-terminal extension comprises multiple threonine residues. In some embodiments, the C-terminal extension comprises multiple histidine residues. In some embodiments, the C-terminal extension comprises multiple glycine residues. In some embodiments, the C-terminal extension comprises multiple proline residues.

[0175] Table 2. Exemplary VHH antibody sequences with extensions. Extensions are shown in bold.

[0176]

[0177] In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 9. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 10. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 11. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 13. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 14. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 15. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 16. In some embodiments, the VHH antibody with C-terminal extension comprises SEQ ID NO: 17.

[0178] In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 85% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 87% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 90% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 92% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension comprises a sequence having at least 93% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension comprises an additional mutation. In some embodiments, the VHH antibody with C-terminal extension comprises a sequence having at least 94% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension comprises an additional mutation. In some embodiments, the VHH antibody with C-terminal extension comprises a sequence having at least 95% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension comprises an additional mutation. In some embodiments, the VHH antibody with C-terminal extension comprises a sequence having at least 96% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension comprises an additional mutation. In some embodiments, the VHH antibody with C-terminal extension comprises a sequence having at least 97% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 98% identity with SEQ ID NO: 12. In some embodiments, the VHH antibody with C-terminal extension contains an additional mutation. In some embodiments, the VHH antibody with C-terminal extension contains a sequence having at least 99% identity with SEQ ID NO: 12.

[0179] Other forms of concealment

[0180] In some embodiments, the anti-IL-2 VHH or its antigen-binding fragment (e.g., a CDR sequence) described herein is incorporated into an anti-IL-2 antigen-binding molecule. In some embodiments, the anti-IL-2 VHH or a fragment thereof is incorporated into an anti-IL-2 antigen-binding molecule selected from: Fab, single-chain Fv (scFv), single-domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single-domain antibody-linked Fab (s-Fab), antibody, or combinations thereof.

[0181] In some embodiments, the anti-IL-2 antigen-binding molecule incorporating the anti-IL-2 VHH sequence described herein can be a complete antibody or a binding fragment thereof. In some embodiments, the binding fragment derived from the antibody includes, but is not limited to, Fab, Fab′, F(ab′)2, scFv, and single-chain antibodies. In some embodiments, the anti-IL-2 antigen-binding molecule incorporates the anti-IL-2 VHH sequence described herein or an antigen-binding fragment thereof (e.g., CDR).

[0182] In some implementations, an anti-IL-2 antigen-binding molecule derived from the anti-IL-2 VHH sequence is used as an antibody-based masking portion of the masked cytokine.

[0183] In some embodiments, the masking portion based on the anti-IL-2 antibody comprises a Fab, 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 Fab (s-Fab) linked to a single-domain antibody, an antibody, or a combination thereof. In some embodiments, the masking portion based on the anti-IL-2 antibody is an scFv. In some embodiments, the masking portion based on the anti-IL-2 antibody is a Fab fragment. In some embodiments, the masking portion based on the anti-IL-2 antibody comprises one or more CDRs that bind to IL-2 (e.g., the CDRs described herein). In some embodiments, the antibody-based masking portion comprises a variable heavy chain (VH) that binds to IL-2. In some embodiments, the antibody-based masking portion comprises a variable light chain (VL) that binds to IL-2. In some embodiments, the antibody-based masking portion comprises a Fab-like bispecific antibody (bsFab) that binds to IL-2. In some embodiments, the antibody-based masking portion comprises a Fab (s-Fab) linked to a single-domain antibody that binds to IL-2. In some implementations, the antibody-based masking portion comprises an antibody or a fragment thereof that binds to IL-2.

[0184] carrier part

[0185] Long in vivo half-lives are crucial for therapeutic proteins. Unfortunately, cytokines administered to subjects typically have short half-lives because they are usually rapidly eliminated from subjects through mechanisms including renal clearance and endocytic degradation. Therefore, for purposes such as prolonging the in vivo half-life of cytokines, the masked cytokines provided herein incorporate a carrier portion linked to the cytokine or masking portion.

[0186] In some embodiments, the carrier portion is a PEG molecule, albumin, an albumin fragment, and an antibody Fc domain, or an antibody or its antigen-binding fragment. In some embodiments, the carrier portion is an Fc domain. In some embodiments, the carrier portion is a PEG molecule. In some embodiments, the carrier portion is albumin. In some embodiments, the carrier portion is an albumin fragment. In some embodiments, the carrier portion is an antibody or its antigen-binding fragment.

[0187] Fc structural domain

[0188] This document provides an Fc domain for use in masked cytokines or their cleavage products. In some embodiments, for purposes such as prolonging the half-life of the cytokine in vivo, the Fc domain is attached to the cytokine or masking portion in the masked cytokines provided herein.

[0189] As used herein, the term "Fc domain" refers to a polypeptide or fragment derived from an immunoglobulin. An Fc domain can be any antibody or fragment thereof. Fc domains are derived from antibodies or fragments thereof naturally occurring in animals (e.g., mammals) or prepared using any techniques known in the art. In some embodiments, the Fc domain is a human Fc domain.

[0190] In some embodiments, the first and second Fc peptides are linked via a linker, such as a short peptide linker. In some embodiments, the linker is non-cleavable.

[0191] In some implementations, the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide.

[0192] 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 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 capable of FcRn-mediated recycling (e.g., an Fc domain or fragment thereof).

[0193] The Fc domain or a fragment thereof can be 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 (such as by including a heavy chain polypeptide). It is recognized in the art that FcRn-mediated recycling requires the binding of the Fc region of the FcRn receptor to the antibody or a fragment thereof. For example, studies have shown that residues I253, S254, H435, and Y436 (numbered according to the Kabat EU indexing system) are crucial 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 indexing system) have also been examined and reported. Yeung, YA et al., (J. Immunol. 182 (2009) 7667-7671).

[0194] In some embodiments, the antibody or a fragment thereof comprises a heavy-chain polypeptide or a light-chain polypeptide. In some embodiments, the antibody or a fragment thereof 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 antibody or a fragment thereof comprises CH2 and CH3 domains or fragments thereof. In some embodiments, the antibody or a fragment thereof comprises a constant domain of a heavy-chain polypeptide. In some embodiments, the antibody or a fragment thereof comprises a constant domain of a light-chain polypeptide. In some embodiments, the antibody or a fragment thereof comprises a heavy-chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). In some embodiments, the antibody or a fragment thereof comprises a light-chain polypeptide.

[0195] In some embodiments, the first Fc domain and / or the second Fc domain each contain one or more modifications that promote non-covalent association between the first Fc peptide and the second Fc peptide. In some embodiments, the first Fc peptide comprises an IgG1 Fc domain or a fragment thereof comprising mutations in Y349C, T366S, L368A, and Y407V to form a 'pound' in the first half-life extension domain, and the second Fc peptide comprises an IgG1 Fc domain or a fragment thereof comprising mutations in S354C and T366W to form a 'mortar' in the second half-life extension domain.

[0196] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are each an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof. In some embodiments, the first Fc polypeptide and the second Fc polypeptide are each an IgG1 Fc domain or a fragment thereof. Human IgG1 immunoglobulin weight constant γ1 has the following sequence:

[0197] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 18)

[0198] In some embodiments, the first Fc polypeptide and the second Fc polypeptide are derived from the sequence of human IgG1 immunoglobulin recurrent γ1 having SEQ ID NO: 18 ('parental sequence'), such that the first Fc polypeptide and the second Fc polypeptide each comprise SEQ ID NO: 18 or a fragment thereof having one or more amino acid modifications.

[0199] In some embodiments, the first Fc polypeptide and the Fc polypeptide each comprise the portion of SEQ ID NO: 18 shown above, optionally having one or more amino acid modifications, namely:

[0200] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 19)

[0201] In some embodiments, the first Fc polypeptide comprises an amino acid sequence having one or more modifications, such as substitutions, additions, or deletions of one or more amino acids, compared to the amino acid sequence of any of SEQ ID NO: 19. In some embodiments, the second Fc polypeptide comprises an amino acid sequence having one or more modifications, such as substitutions, additions, or deletions of one or more amino acids, compared to the amino acid sequence of any of SEQ ID NO: 19. 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.

[0202] In some embodiments, the Fc domain or a fragment thereof contains one or more amino acid substitutions that alter the effector function. In some embodiments, the half-life extension domain is the IgG1 Fc domain or a fragment thereof, and contains 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, which are 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 amino substitutions: V234A and G237A; H268Q, V309L, A330S, and A331S; and / or V234A, G237A, P238S, H268A, V309L, and A330S, which are 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, which are 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 amino substitutions: L235A, G237A, and E318A; S228P, L234A, and L235A; H268Q, V309L, A330S, and P331S; and / or S228P and L235A, which are 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, which are numbered according to the Kabat EU numbering system.

[0203] In some embodiments, the Fc domain or a fragment thereof contains one or more amino acid substitutions that enhance the effector function. In some embodiments, the half-life extension domain is IgG1. The Fc domain or a fragment thereof, and containing the following 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 E33 3S; K290E, S298G and T299A; K290E, S298G, T299A and K326E; K290N, S298G and T299A; K290N, S298G, T299A and K326E; K334V; L235S, S239D and K334V; K334V and Q331M, S239D, F243V, E294L or S298T; E233L, Q311M and K334V; L234I, Q311M and K334V; K334V and S298T, A330M or A330F; K334V, Q311M and A330M or A330F; K334 V, 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; L328I and I332E; L328Q and I332E; V264T V240I; V266I; S239D; S239D and I332D; S239D and I332N; S239D and I332Q; S239E and I332D; S239E and I332N; S239E and I332Q; S239N and I332D; S239N and I332E; S239Q and I332D; A330Y and I332E; V264I, A330Y and I332E; A330L and I332E; V264I, A330L and I332E; L234E, L234Y or L234I; L235D, L235S, L235Y or L235I; S239T;V240M; V264Y; A330I; N325T; I332E and L328D, L328V, L328T or L328I; V264I, I332E and S239E or S239Q; S239E, V264I, A330Y and I332E; A330Y, I332E and S239D or S239N; A330L, I332E and S2 39D 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 V2 66I; 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, which are numbered according to the Kabat EU numbering system. In some embodiments, the 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.

[0204] In some embodiments, the Fc domain includes 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 an Fc domain or fragment thereof) to FcRn at acidic pH. In some embodiments, the half-life extension domain comprises one or more amino acid substitutions selected from the group consisting of: M428F; T250Q and M428F; M252Y, S254T and T256E; P257I and N434H; D376V and N434H; P257I and Q3111; N434A; N434W; M428F and N434S; V259I and V308F; M252Y, S254T and T256E; V259I, V308F and M428F; T307Q and N434A; T307Q and N434S; T307Q, E380A and N434A; V308P and N434A; N434H; and V308P.

[0205] Mortar and pestle method

[0206] One strategy for promoting the heterodimerization of two Fc peptides is a method called the "mortar and pestle".

[0207] In some embodiments, the targeted cytokine comprises a first Fc polypeptide and a second Fc polypeptide, each containing a CH3 domain. In some embodiments, the Fc polypeptide containing the CH3 domain is a heavy chain polypeptide or a fragment thereof (e.g., an Fc domain or a fragment thereof). The CH3 domains of the two Fc polypeptides can be altered using a "mortar and pestle" technique, which is described in detail in several examples, such as WO1996 / 027011; Ridgway, JB et al., Protein Eng. (1996) 9(7): 617-621; Merchant, AM et al., Nat. Biotechnol. (1998) 16(7): 677-681. See also Klein et al. (2012), MAbs, 4(6): 653-663. Using the mortar and pestle method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of the two half-life-extending domains containing the two altered CH3 domains. This is achieved by introducing a bulky residue into a CH3 domain that has a long half-life, which acts as a "pestle". Then, to accommodate the bulky residue, a "mortar" is formed in another long half-life domain that can accommodate the pestle. Either altered CH3 domain can be a "pestle" and the other can be a "mortar". The introduction of disulfide bonds further stabilizes the heterodimer (Merchant, AM et al., Nat. Biotechnol. (1998) 16(7); Atwell, S. et al., J. Mol.Biol. (1997) 270(1): 26-35) and increases the yield.

[0208] It has been reported that a heterodimerization yield of over 97% can be achieved by introducing the S354C and T366W mutations into the heavy chain to produce a "pestle," and by introducing the Y349C, T366S, L368A, and Y407V mutations into the heavy chain to produce a "mortar" (residues numbered according to the KabatEU numbering system). Carter et al. (2001), J. Immunol. Methods, 248:7-15; Klein et al. (2012), MAbs, 4(6): 653-663.

[0209] In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first half-life Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system). In some embodiments comprising a first Fc polypeptide and a second Fc polypeptide, the first Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU numbering system), and the second Fc polypeptide comprises a heavy chain polypeptide or a portion thereof (e.g., an Fc domain or a fragment thereof) containing amino acid mutations S354C and T366W (numbered according to the Kabat EU numbering system).

[0210] Further examples of substitutions that can be made to form pestles and mortars include those described in US20140302037A1, the contents of which are incorporated herein by reference. For example, in some embodiments, the first Fc polypeptide (“first domain”) and the paired second Fc polypeptide (“second domain”), each containing an Fc domain, can be substituted with any of the following amino acid substitutions: (a) Y407T in the first domain and T366Y in the second domain; (b) Y407A in the first domain and T366W in the second domain; (c) F405A in the first domain and T394W in the second domain; (d) F405W in the first domain and T394S in the second domain; (e) the first domain (a) Y407T in the first domain and T366Y in the second domain; (b) T366Y and F405A in the first domain and T394W and Y407T in the second domain; (c) T366W and F405W in the first domain and T394S and Y407A in the second domain; (d) F405W and Y407A in the first domain and T366W and T394S in the second domain; or (i) T366W in the first domain and T366S, L368A and Y407V in the second domain, which are numbered according to the Kabat EU numbering system.

[0211] In some embodiments, the first Fc polypeptide (“first domain”) and the paired second Fc polypeptide (“second domain”), each containing an Fc domain, may be substituted with any of the following amino acid substitutions: (a) Y407T in the second domain and T366Y in the first domain; (b) Y407A in the second domain and T366W in the first domain; (c) F405A in the second domain and T394W in the first domain; (d) F405W in the second domain and T394S in the first domain; (e) [missing information - likely a specific amino acid substitution]. Y407T and T366Y in the first structural domain; (f) T366Y and F405A in the second structural domain and T394W and Y407T in the first structural domain; (g) T366W and F405W in the second structural domain and T394S and Y407A in the first structural domain; (h) F405W and Y407A in the second structural domain and T366W and T394S in the first structural domain; or (i) T366W in the second structural domain and T366S, L368A and Y407V in the first structural domain, which are numbered according to the Kabat EU numbering system.

[0212] In embodiments comprising a first Fc polypeptide and a second Fc polypeptide, each containing an Fc domain, any of the heterodimerization modifications described herein can be applied to the Fc domain to promote heterodimerization of any of the targeted cytokines described herein.

[0213] In some embodiments, the first Fc polypeptide and the second Fc polypeptide comprise SEQ ID NO: 19 having amino substitutions for promoting association between the first Fc polypeptide and the second Fc polypeptide according to the 'knobinto holes' method. In some embodiments, sequence SEQ ID NO: 19 contains mutations Y349C; T366S; L368A; and Y407V (numbered according to the Kabat EU numbering system) to form 'mortars' in the first Fc polypeptide, and mutations S354C and T366W (numbered according to the Kabat EU numbering system) to form 'knobinto holes' in the second Fc polypeptide.

[0214] In some embodiments, the first Fc polypeptide comprises the mutations Y349C, T366S, L368A, and Y407V relative to SEQ ID NO: 19. In some embodiments, the second Fc polypeptide comprises the mutations S354C and T366W relative to SEQ ID NO: 19.

[0215] In some embodiments, the first and second half-life extension domains each further comprise an amino-substituted N297A numbered according to the Kabat EU numbering system. In some embodiments, the first Fc polypeptide comprises the mutations Y349C, T366S, L368A, Y407V, and N297A relative to SEQ ID NO: 19. In some embodiments, the second Fc polypeptide comprises the mutations S354C, T366W, and N297A relative to SEQ ID NO: 19.

[0216] First Fc polypeptide (Y349C; T366S; L368A; Y407V and N297A) SEQ ID NO: 20:

[0217] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20)

[0218] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each further comprise an amino-substituted I253A numbered according to the Kabat EU numbering system.

[0219] In some embodiments, the first Fc polypeptide and the second Fc polypeptide each further comprise amino-substituted N297A and I253A, numbered according to the Kabat EU numbering system. In some embodiments, the first Fc polypeptide comprises the mutants Y349C, T366S, L368A, Y407V, N297A, and I253A relative to SEQ ID NO: 19. In some embodiments, the second Fc polypeptide comprises the mutants S354C, T366W, N297A, and I253A relative to SEQ ID NO: 19.

[0220] RF mutations or CH3 domain exchanges used for the purification of heterodimeric proteins

[0221] Two immunoglobulin heavy chains differing by at least one amino acid allow for the separation of antigen-binding proteins based on the different affinities of the immunoglobulin heavy chain 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 allow for rapid separation via differential binding of the IgG region to protein A.

[0222] In one embodiment, the second Fc polypeptide includes a 95R modification (according to IMGT exon numbering; 435R according to EU numbering) in the CH3 region. In another embodiment, the second Fc polypeptide also includes a 96F modification (IMGT; 436F according to EU numbering). In some embodiments, the first Fc polypeptide includes wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide includes a 95R / 96F modification according to IMGT exon numbering. In some embodiments, the first Fc polypeptide includes wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide includes a 435R / 436F modification according to EU numbering.

[0223] In some embodiments, the first Fc polypeptide comprises wild-type CH2 and CH3 domains derived from IgG1 or IgG4, and the second Fc polypeptide comprises a CH3 domain derived from IgG3.

[0224] In some embodiments, the first Fc polypeptide or the second Fc polypeptide comprises the amino acid sequence of SEQ ID NO: 21. SEQ ID NO: 21 comprises a "pellet mutation" having an "RF mutation (435R / 436F). In some embodiments, the first Fc polypeptide comprises mutations of S354C, T366W, N297A, H435R, and Y435F relative to SEQ ID NO: 19.

[0225] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRFTQKSLSLSPGGSPG (SEQ ID NO: 21)

[0226] 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 any of the amino acid sequences of SEQ ID NO: 19 or SEQ ID NO: 20.

[0227] In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 87% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 92% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 93% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 94% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 96% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 97% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 19.

[0228] In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 87% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 92% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 93% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 94% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 96% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 97% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 20.

[0229] In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 85% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 87% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 92% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 93% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 94% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 95% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 96% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 97% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 98% sequence identity with the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first Fc polypeptide or the second polypeptide comprises an amino acid sequence having about or at least about 99% sequence identity with the amino acid sequence of SEQ ID NO: 21.

[0230] In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 20, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the first polypeptide comprises the amino acid sequence of SEQ ID NO: 21, and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 20.

[0231] connector

[0232] This article provides adapters for use in masked cytokines or their cleavage products. As provided herein, adapters refer to peptides having two or more amino acids that are used to link two functional components together in the masked cytokines described herein.

[0233] In some embodiments, the masked cytokine comprises a first connector and a second connector, wherein at least the first connector or the second connector comprises a proteolytically cleaved peptide. In some embodiments, the masked cytokine comprises a first connector and a second connector, wherein both the first connector and the second connector are non-cleavable connectors.

[0234] In some embodiments, a first Fc polypeptide is linked to a cytokine or a variant thereof via a first adapter. In some embodiments, a second Fc polypeptide is linked to a masking portion via a second adapter. In some embodiments, the first Fc polypeptide is linked to a cytokine or a variant thereof via a cleavable adapter. In some embodiments, the second Fc polypeptide is linked to a masking portion via a cleavable adapter. In some embodiments, the first Fc polypeptide is linked to a cytokine or a variant thereof via a cleavable adapter, and the second Fc polypeptide is linked to a masking portion via a non-cleavable adapter. In some embodiments, the first Fc polypeptide is linked to a cytokine or a variant thereof via a non-cleavable adapter, and the second Fc polypeptide is linked to a masking portion via a cleavable adapter. In some embodiments, the first Fc polypeptide is linked to a cytokine or a variant thereof via a cleavable adapter, and the second Fc polypeptide is linked to a masking portion via a non-cleavable adapter.

[0235] Cuttable connector

[0236] The cuttable connector in this invention contains a cutting peptide.

[0237] Cleavage peptides

[0238] A cleavage peptide is a polypeptide that includes a protease cleavage site, allowing it to be hydrolyzed by proteolysis. A protease is an enzyme that cleaves and hydrolyzes the peptide bond between two specific amino acid residues of a target substrate protein. As used herein, a “cleavage site” refers to a recognizable site for cleaving a portion of the cleavage peptide present in any linker comprising the cleavage peptide described herein. Thus, a cleavage site can be present in the cleavage peptide sequence as described herein. In the context of this disclosure, the terms “cleavage peptide” and “cleavage peptide motif” are used interchangeably. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleaving agent.

[0239] In some implementations, cleavage sites may be incorporated into the adapter, which is used to link two functional components together in the masked cytokine described herein.

[0240] In some implementations, the protease cleavage site is a tumor-associated protease cleavage site. As used herein, a "tumor-associated protease cleavage site" is an amino acid sequence recognized by a protease whose expression is specific to or upregulated in relation to tumor cells or their tumor cell environment.

[0241] The tumor cell environment is complex and can contain a variety of different proteases. Therefore, the precise site at which a given cleavage peptide will be cleaved in the tumor cell environment can vary between tumor types, between patients with the same tumor type, and even between the cleavage products formed in the same tumor, depending on the specific tumor cell environment. Furthermore, even after cleavage, further modifications to the initial cleavage product (e.g., by removing one or two terminal amino acids) can occur due to further action by proteases in the tumor cell environment. Therefore, after administration of a single-structure, targeted cytokine as described herein, the distribution of the cleavage product in the patient's tumor cell environment can be expected.

[0242] It should be understood that the cleavage site mentioned herein refers to the site between two specific amino acid residues within a cleavage peptide, which are known targets of proteases associated with the tumor cell environment. In this sense, a cleavage peptide as described herein may contain more than one cleavage site, with different proteases cleaving the peptide at different cleavage sites. It is also possible that more than one protease can act on the same cleavage site within the cleavage peptide. Discussions of protease cleavage sites can be found in the art.

[0243] Therefore, the cleavage peptides disclosed herein can be cleaved by one or more proteases. In some embodiments, the protease cleavage site is a tumor-associated protease cleavage site. The tumor-associated protease cleavage site can be recognized by the tumor-associated protease. As a non-limiting example, 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 some embodiments, the tumor-associated protease is MMP7. In another embodiment, the tumor-associated protease is MMP9. In another embodiment, the tumor-associated protease is MMP10. Other disease-related and tissue-selective proteases include, but are not limited to, cathepsins (catB, cathepsin D, cathepsin F, cathepsin K, cathepsin L, cathepsin V, cathepsin S, 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 activating factor (uPA), trypsin, caspase, thrombin, legumin, chymase, collagenase, aspartic protease A (napsin A), and matripatse 1-2. In some embodiments, the disease-related and / or tissue-selective protease is cathepsin (catB).

[0244] In some implementations, the cleavage peptide is a substrate of a protease co-localized in a region or tissue expressing a cytokine receptor.

[0245] In some embodiments, the cleaved peptide is a pentamelider (i.e., a peptide with a length of 5 amino acids), a hexamer (i.e., a peptide with a length of 6 amino acids), a heptamer (i.e., a peptide with a length of 7 amino acids), an octamer (i.e., a peptide with a length of 8 amino acids), a nonamelider (i.e., a peptide with a length of 9 amino acids), a decamer (i.e., a peptide with a length of 10 amino acids), an 11-mer (i.e., a peptide with a length of 11 amino acids), a 12-mer (i.e., a peptide with a length of 12 amino acids), a 13-mer (i.e., a peptide with a length of 13 amino acids), a 14-mer (i.e., a peptide with a length of 14 amino acids), a 15-mer (i.e., a peptide with a length of 15 amino acids), a 16-mer (i.e., a peptide with a length of 16 amino acids), a 17-mer (i.e., a peptide with a length of 17 amino acids), or an octamer (i.e., a peptide with a length of 18 amino acids).

[0246] In some embodiments, the length of the cleaved peptide is 5 to 18 amino acids. In some embodiments, the length of the cleaved peptide is 6 to 10 amino acids.

[0247] In some embodiments, the cleaving peptide comprises SEQ ID NO: 22 (IHVTLKSL). In some embodiments, the cleaving peptide comprises SEQ ID NO: 23 (NSYTIKGL). In some embodiments, the cleaving peptide comprises SEQ ID NO: 24 (SNESLSLS). In some embodiments, the cleaving peptide comprises SEQ ID NO: 25 (QVSSSLSP). In some embodiments, the cleaving peptide comprises SEQ ID NO: 26 (PTSTSLSP). In some embodiments, the cleaving peptide comprises SEQ ID NO: 27 (ESLSLSEE). In some embodiments, the cleaving peptide comprises SEQ ID NO: 28 (ASLSLAPV). In some embodiments, the cleaving peptide comprises SEQ ID NO: 29 (SQESLSLS). In some embodiments, the cleaving peptide comprises SEQ ID NO: 30 (PLGL). In some embodiments, the cleaving peptide comprises SEQ ID NO: 31 (APAGLIVPYN). In some embodiments, the cleaving peptide comprises SEQ ID NO: 32 (PVSLRSGS). In some embodiments, the cleaving peptide comprises PANLVAPDP (SEQ ID NO: 33). In some embodiments, the cleaving peptide comprises MPYDLYHP (SEQ ID NO: 34). In some embodiments, the cleaving peptide comprises ISSGLLSGRS (SEQ ID NO: 35). In some embodiments, the cleaving peptide comprises RAAAVKSP (SEQ ID NO: 36). In some embodiments, the cleaving peptide comprises SEQ ID NO: 37 (RPLALWRS). In some embodiments, the cleaving peptide comprises SEQ ID NO: 38 (TQKPLGLS). In some embodiments, the cleaving peptide comprises SEQ ID NO: 39 (RSKYLATA). In some embodiments, the cleaving peptide comprises GRPRHQGV (SEQ ID NO: 40). In some embodiments, the cleaving peptide comprises GLFG (SEQ ID NO: 41). In some embodiments, the cleaving peptide comprises GFLG (SEQ ID NO: 42). In some embodiments, the cleaving peptide comprises AGRRAAK (SEQ ID NO: 43). In some embodiments, the cleaving peptide comprises FRLWA (SEQ ID NO: 44). In some embodiments, the cleaving peptide comprises FRLWS (SEQ ID NO: 45). In some embodiments, the cleavage peptide comprises NFFGVGGE (SEQ ID NO: 46). In some embodiments, the cleavage peptide comprises PMKRLTLA (SEQ ID NO: 47).In some embodiments, the cleaving peptide comprises FPLATYAP (SEQ ID NO: 48). In some embodiments, the cleaving peptide comprises FLVGGASL (SEQ ID NO: 49). In some embodiments, the cleaving peptide comprises KPMQFLGD (SEQ ID NO: 50). In some embodiments, the cleaving peptide comprises GIVRAKGV (SEQ ID NO: 51). In some embodiments, the cleaving peptide comprises ALFKSSFP (SEQ ID NO: 52). In some embodiments, the cleaving peptide comprises SGRRSPGG (SEQ ID NO: 53). In some embodiments, the cleaving peptide comprises SLGRRPGG (SEQ ID NO: 54). In some embodiments, the cleaving peptide comprises SLSGRRGG (SEQ ID NO: 55). In some embodiments, the cleaving peptide comprises SLSLGRRG (SEQ ID NO: 56). In some embodiments, the cleaving peptide comprises SLSLSGRR (SEQ ID NO: 57). In some embodiments, the cleaving peptide comprises GGPRRL (SEQ ID NO: 58). In some embodiments, the cleaving peptide comprises GGPLRL (SEQ ID NO: 59). In some embodiments, the cleaving peptide comprises GGPKLL (SEQ ID NO: 60). In some embodiments, the cleaving peptide comprises GGPRNL (SEQ ID NO: 61). In some embodiments, the cleaving peptide comprises GGPRML (SEQ ID NO: 62). In some embodiments, the cleaving peptide comprises EHLRSPGG (SEQ ID NO: 63). In some embodiments, the cleaving peptide comprises FRSGVPGG (SEQ ID NO: 64). In some embodiments, the cleaving peptide comprises SLLLRTGN (SEQ ID NO: 65). In some embodiments, the cleaving peptide comprises AGLRSPGG (SEQ ID NO: 66). In some embodiments, the cleaving peptide comprises SLFRSAGP (SEQ ID NO: 67). In some embodiments, the cleaving peptide comprises SLFRAPGP (SEQ ID NO: 68). In some embodiments, the cleaving peptide comprises WLFRSPLG (SEQ ID NO: 69). In some embodiments, the cleaving peptide comprises SRLRSPQG (SEQ ID NO: 70). In some embodiments, the cleavage peptide comprises SLVLSGRR (SEQ ID NO: 71). In some embodiments, the cleavage peptide comprises KQLRHMRG (SEQ ID NO: 72). In some embodiments, the cleavage peptide comprises LSGRSDNH (SEQ ID NO: 73).In some embodiments, the cleaving peptide comprises LSGK (SEQ ID NO: 74). In some embodiments, the cleaving peptide comprises LSGR (SEQ ID NO: 75). In some embodiments, the cleaving peptide comprises RQARVVGG (SEQ ID NO: 76). In some embodiments, the cleaving peptide comprises RQRRVVGG (SEQ ID NO: 77). In some embodiments, the cleaving peptide comprises RQYRVVGG (SEQ ID NO: 78). In some embodiments, the cleaving peptide comprises SKGRSLIG (SEQ ID NO: 79). In some embodiments, the cleaving peptide comprises PRFKIIGG (SEQ ID NO: 80). In some embodiments, the cleaving peptide comprises KQLRVVNG (SEQ ID NO: 81). In some embodiments, the cleaving peptide comprises IQPRITGG (SEQ ID NO: 82). In some embodiments, the cleaving peptide comprises KQSRKFVP (SEQ ID NO: 83). In some embodiments, the cleaving peptide comprises GRQSRAGG (SEQ ID NO: 84). In some embodiments, the cleaving peptide comprises SGRSSPGG (SEQ ID NO: 85). In some embodiments, the cleaving peptide comprises SSGRSPGG (SEQ ID NO: 86). In some embodiments, the cleaving peptide comprises SLSGRSGG (SEQ ID NO: 87). In some embodiments, the cleaving peptide comprises SLSSGRSG (SEQ ID NO: 88). In some embodiments, the cleaving peptide comprises KLSLSGRS (SEQ ID NO: 89). In some embodiments, the cleaving peptide comprises PLRLSRA (SEQ ID NO: 90). In some embodiments, the cleaving peptide comprises PLKLSRA (SEQ ID NO: 91). In some embodiments, the cleaving peptide comprises PLGLSGRS (SEQ ID NO: 92). In some embodiments, the cleaving peptide comprises PLGLRSRA (SEQ ID NO: 93). In some embodiments, the cleaving peptide comprises PLGLKSRA (SEQ ID NO: 94). In some embodiments, the cleaving peptide comprises RGSRAG (SEQ ID NO: 95). In some embodiments, the cleaving peptide comprises RLSRGK (SEQ ID NO: 96). In some embodiments, the cleavage peptide comprises RGSRGGG (SEQ ID NO: 97). In some embodiments, the cleavage peptide comprises KGSRAG (SEQ ID NO: 98). In some embodiments, the cleavage peptide comprises KLSRGK (SEQ ID NO: 99).In some embodiments, the cleaving peptide comprises GRSRAG (SEQ ID NO: 100). In some embodiments, the cleaving peptide comprises LRSRGK (SEQ ID NO: 101). In some embodiments, the cleaving peptide comprises GRSRGG (SEQ ID NO: 102). In some embodiments, the cleaving peptide comprises GKSRAG (SEQ ID NO: 103). In some embodiments, the cleaving peptide comprises LKSRGK (SEQ ID NO: 104). In some embodiments, the cleaving peptide comprises VPLSLYSGP (SEQ ID NO: 105). In some embodiments, the cleaving peptide comprises DSGGFMLT (SEQ ID NO: 106). In some embodiments, the cleaving peptide comprises HEQLTV (SEQ ID NO: 107). In some embodiments, the cleaving peptide comprises VPLSLY (SEQ ID NO: 108). In some embodiments, the cleaving peptide comprises DLLAVVAAS (SEQ ID NO: 109). In some embodiments, the cleaving peptide comprises GMPKDLYHAS (SEQ ID NO: 110). In some embodiments, the cleaving peptide comprises IVGRPRHQGV (SEQ ID NO: 111). In some embodiments, the cleaving peptide comprises MPYDLYHPS (SEQ ID NO: 112). In some embodiments, the cleaving peptide comprises VPLSLYSG (SEQ ID NO: 113). In some embodiments, the cleaving peptide comprises ISSGLLSGRSDQP (SEQ ID NO: 114). In some embodiments, the cleaving peptide comprises MPY (SEQ ID NO: 115). In some embodiments, the cleaving peptide comprises QQGNVFSC (SEQ ID NO: 116). In some embodiments, the cleaving peptide comprises LLSSGRS (SEQ ID NO: 117).

[0248] Table 3. Exemplary cleavage peptides

[0249]

[0250]

[0251]

[0252] By way of example only, in the table above, * indicates a known or observed protease cleavage site in the cleavage peptide.

[0253] In some implementations, the cleavage peptide comprises an amino acid sequence selected from Table 3.

[0254] In some embodiments, the length of the cutterable linker is between 2 and 25 amino acids. In some embodiments, the length of the cutterable linker is between 3 and 21 amino acids. In some embodiments, the length of the cutterable linker is between 3 and 18 amino acids. In some embodiments, the length of the cutterable linker is between 5 and 18 amino acids. In some embodiments, the length of the cutterable linker is between 3 and 8 amino acids. In some embodiments, the length of the cutterable linker is between 4 and 6 amino acids.

[0255] In some embodiments, the length of the cuttable linker is 15 amino acids. In some embodiments, the length of the cuttable linker is 16 amino acids. In some embodiments, the length of the cuttable linker is 17 amino acids. In some embodiments, the length of the cuttable linker is 18 amino acids. In some embodiments, the length of the cuttable linker is 19 amino acids. In some embodiments, the length of the cuttable linker is 20 amino acids.

[0256] In some implementations, the cleavable connector comprises the cleaving peptides listed in Table 3.

[0257] In some embodiments, the cleavable linker comprises a proteolytic cleavage peptide (CP) flanked by spacer domains (SD), as shown in the following formula:

[0258] SD-CP-SD

[0259] The cleavable connector contains any cleavage peptide selected from Table 3.

[0260] In some embodiments, the cuttable connector comprises MPYDLYHPS (SEQ ID NO: 112). In some embodiments, the cuttable connector comprises MPY (SEQ ID NO: 115).

[0261] Table 4. Exemplary Cuttable Connectors

[0262]

[0263] In some implementations, the cuttable connector comprises SPGGGGPMPYDLYHPSGGG (SEQ ID NO: 144).

[0264] Spacing domain

[0265] The spacer domain may consist of one or more amino acids. The function of the spacer domain (where present) is to link the proteolytically cleaved peptide (CP) to other functional components within the construct described herein.

[0266] It should be understood that the spacer domain does not alter the biological interaction between the proteolytic cleavage peptide and proteases in either the tumor cell environment or the non-tumor cell environment. In other words, even in the presence of the spacer domain, the proteolytic cleavage peptides of the present invention disclosed herein retain their advantageous tumor specificity.

[0267] In some implementations, the spacer domains of the lateral proteolytic cleavage peptides differ.

[0268] In some implementations, the spacer domain is rich in amino acid residues G, S, and P.

[0269] In some implementations, the spacer domain includes only amino acid residue types selected from the group consisting of G, S, and P.

[0270] In some implementations, the cutable connector includes type 12:

[0271] N' SD1-CP-SD2 C' (12)

[0272] SD1 is the first spaced structure domain and SD2 is the second spaced structure domain.

[0273] In some implementations, the cutable connector includes type 12:

[0274] N' SD1-CP-SD2 C' (12)

[0275] In some embodiments, the first polypeptide chain comprises formula 7 and the second polypeptide chain comprises formula 13:

[0276] N' HL1 - Uncuttable L1-MM C' (7)

[0277] N' HL2- SD1-CP-SD2 -C' (13)

[0278] In some embodiments, the first polypeptide chain comprises formula 14 and the second polypeptide chain comprises formula 10:

[0279] N' HL1- SD1-CP-SD2 -MM C' (14)

[0280] N' HL2-Indivisible L2-C C' (10)

[0281] In some implementations, SD1 is composed of glycine (G).

[0282] In some implementations, the N-terminus of SD1 is glycine (G).

[0283] In some embodiments, the length of the first spacer domain (SD1) is between 3 and 10 amino acids. In some embodiments, the length of the first spacer domain (SD1) is between 4 and 9 amino acids. In some embodiments, the length of the first spacer domain (SD1) is between 3 and 6 amino acids.

[0284] In some embodiments, SD1 comprises a sequence selected from SEQ ID NO: 145-161. In some embodiments, SD2 comprises a sequence selected from SEQ ID NO: 145-161.

[0285] In some implementations, SD2 consists of GP.

[0286] In some implementations, the C-terminal sequence of SD2 is –GP C'.

[0287] In some implementations, the length of the second spacer domain (SD2) is between 3 and 6 amino acids.

[0288] Table 5. Exemplary Spacing Domains

[0289]

[0290] Table 6 shows an exemplary combination of SD1 and SD2 in a cuttable connector.

[0291] Table 6. Cuttable Joint Structure

[0292]

[0293] Uncuttable connector

[0294] In some embodiments, the length of the uncuttable linker is between 2 and 25 amino acids. In some embodiments, the length of the uncuttable linker is between 3 and 21 amino acids. In some embodiments, the length of the uncuttable linker is between 3 and 18 amino acids. In some embodiments, the length of the uncuttable linker is between 5 and 18 amino acids. In some embodiments, the length of the uncuttable linker is between 3 and 8 amino acids. In some embodiments, the length of the uncuttable linker is between 4 and 6 amino acids.

[0295] In some embodiments, the uncuttable linker is 15 amino acids long. In some embodiments, the uncuttable linker is 16 amino acids long. In some embodiments, the uncuttable linker is 17 amino acids long. In some embodiments, the uncuttable linker is 18 amino acids long. In some embodiments, the uncuttable linker is 19 amino acids long. In some embodiments, the uncuttable linker is 20 amino acids long.

[0296] In some embodiments, the uncleavable linker is rich in amino acid residues G, S, and P. In some embodiments, the uncleavable linker includes only amino acid residue types selected from the group consisting of G, S, and P. In some embodiments, the uncleavable linker includes a 'GS' repeating sequence. In some embodiments, the uncleavable linker includes an N'-terminal 'P' residue.

[0297] In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 162 (PGSGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 163 (GGSSPPGGGSSGGGSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 164 (GGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 165 (GGGGSGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 166 (GGSGGGSGGGGGS). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 167 (GGSGGSGGSGGSGGSSGP). In some embodiments, the non-cleavable linker comprises the amino acid sequence shown in SEQ ID NO: 168 (PGGSGP).

[0298] In some implementations, the non-cleavable linker contains the amino acid sequence GGS.

[0299] In some implementations, the non-cuttable joint includes [(G)] n S], where n = 4 or 5.

[0300] In some embodiments, it is desirable that the first and second polypeptide chains have the same or similar lengths to facilitate the association of the first half-life extension domain with the second half-life extension domain and to facilitate the masking portion masking cytokines or functional fragments thereof in the assembled construct. Therefore, when the masking portion is a shorter amino acid sequence than the cytokine or its functional fragment, the difference in length can be fully or partially compensated for by using a longer linker L1.

[0301] Targeted portion

[0302] In some embodiments, the masked IL-2 cytokine described herein further comprises a targeting portion (e.g., a targeted cytokine). Therefore, the present invention also provides a targeted cytokine comprising a targeting portion, a cytokine or a variant thereof (e.g., an IL-2 peptide), a masking portion (e.g., the anti-IL-2 VHH described herein), and a carrier portion. In some embodiments, the targeted cytokine comprises a targeting portion, an IL-2 peptide, a masking portion, and an Fc domain. The targeted cytokine of the present invention becomes active at the disease site and can specifically target cells of interest for effective cancer treatment without causing undesirable side effects.

[0303] The targeted IL-2 peptide according to this disclosure can bind to the IL-2 peptide or a functional fragment thereof as described anywhere herein; the masking portion as described anywhere herein; the first Fc domain and the second Fc domain as described anywhere herein; the cleavable and non-cleavable linkers as described anywhere herein; and the targeting portion as described anywhere herein.

[0304] This article provides a targeted cytokine comprising a targeting portion. In some embodiments, the targeting portion includes an antigen-binding portion that binds to an antigen expressed on the surface of a target cell.

[0305] 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.

[0306] 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), Exonucleotide 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), Sealin 18.2, Sealin 6, 5T4, Fibroblast Activating Protein α (FAPα), fibronectin, melanoma-associated chondroitin sulfate proteoglycan (MCSP), epithelial cell adhesion molecule (EPCAM), or combinations thereof.

[0307] 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.

[0308] PD-1 targeting portion

[0309] 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.

[0310] 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.

[0311] In some implementations, the target portion binds to PD-L1.

[0312] In some implementations, the targeting portion includes a reagent, peptide, or polypeptide that binds specifically to the target.

[0313] 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.

[0314] In some implementations, the targeting portion includes the heavy chain variable region or the light chain variable region of pembrolizumab.

[0315] In some implementations, the targeting portion includes the heavy chain variable region of QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS (SEQ ID NO: 169).

[0316] 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: 169. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO: 169.

[0317] In some implementations, the targeting portion comprises the following heavy chain variable region and constant region: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 170).

[0318] 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: 170. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO: 170.

[0319] In some implementations, the targeting portion includes QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK RVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 171) heavy chain

[0320] 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: 171. In some embodiments, the targeting portion comprises the amino acid sequence of SEQ ID NO: 171.

[0321] In some implementations, the targeting portion includes the heavy chain CDR1 sequence of GYTFTNYY (SEQ ID NO: 172), the heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 173), and the heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 174).

[0322] In some implementations, the targeting portion contains a light chain variable region of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKTSENLYFQ (SEQ ID NO: 175).

[0323] 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: 175. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 175.

[0324] In some implementations, the targeting portion comprises a light chain of EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 176).

[0325] 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: 176. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 176.

[0326] In some implementations, the targeting portion includes the light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 177), the light chain CDR2 sequence of LAS (SEQ ID NO: 178), and the light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 179).

[0327] In some implementations, the target portion includes HCDR1 of SEQ ID NO: 172, HCDR2 of SEQ ID NO: 173, HCDR3 of SEQ ID NO: 174, LCDR1 of SEQ ID NO: 177, LCDR2 of SEQ ID NO: 178, and LCDR3 of SEQ ID NO: 179.

[0328] In some implementations, the targeting portion includes the heavy chain variable region or the light chain variable region of nivolumab.

[0329] In some implementations, the targeting portion includes the heavy chain variable region of QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS (SEQ ID NO: 180).

[0330] 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: 180. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 180.

[0331] In some implementations, the targeting portion includes QVQLVESGGG VVQPGRSLRL DCKASGITFSNSGMHWVRQA PGKGLEWVAV IWYDGSKRYY ADSVKGRFTI SRDNSKNTLF LQMNSLRAED TAVYYCATNDDYWGQGTLVT VSSASTKGPS VFPLAPCSRS TSESTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVLQSSGLYSLSS VVTVPSSSLG TKTYTCNVDH KPSNTKVDKR VESKYGPPCP PCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN STYRVVSVLTVLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE MTKNQVSLTC Heavy chain of LVKGFYPSDIAVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW QEGNVFSCSV MHEALHNHYT QKSLSLSLGK (SEQ ID NO: 181).

[0332] 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: 181. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 181.

[0333] In some implementations, the targeting portion includes the heavy chain CDR1 sequence of GITFSNSG (SEQ ID NO: 182), the heavy chain CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 183), and the heavy chain CDR3 sequence of ATNDDY (SEQ ID NO: 184).

[0334] In some implementations, the targeting portion includes a heavy chain variable region containing SEQ ID NO: 185 and a CH1 domain.

[0335] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 185).

[0336] In some implementations, the targeting portion contains a light chain variable region of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK (SEQ ID NO: 186).

[0337] 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: 186. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 186.

[0338] 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: 187).

[0339] 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: 187. In some embodiments, the targeting portion comprises the same amino acid sequence as SEQ ID NO: 187.

[0340] In some implementations, the targeting portion includes the light chain CDR1 sequence of QSVSSY (SEQ ID NO: 188), the light chain CDR2 sequence of DAS (SEQ ID NO: 189), and the light chain CDR3 sequence of QQSSNWPRT (SEQ ID NO: 190).

[0341] In some embodiments, the targeting portion comprises a heavy chain variable region and a CH1 domain. In some embodiments, the heavy chain variable region and the CH1 domain comprise an amino acid sequence (SEQ ID NO: 185).

[0342] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 185).

[0343] 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.

[0344] 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.

[0345] Exemplary masked forms of cytokines

[0346] The present invention particularly provides a masked cytokine comprising an IL-2 polypeptide or a variant thereof, a masking portion, and a carrier portion. The masked IL-2 cytokine of the present invention becomes active at the tumor site by cleaving a cleaving peptide located within the masked cytokine using a tumor-specific protease, which releases the IL-2 polypeptide upon cleavage.

[0347] 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 a masking portion.

[0348] In particular, the masked cytokine or targeted cytokine of the present invention comprising the VHH masking portion 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).

[0349] In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine, a VHH masking portion, and a vector portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine, a VHH masking portion, and an Fc domain. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing an F42E mutation, a VHH masking portion, and a vector portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing mutations in both F42E and C125A, a VHH masking portion, and a vector portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing mutations in both F42E and C125A, a CDR1 containing GSIFSINVMG (SEQ ID NO: 6), a CDR2 containing AISSGGSTNYADSVKG (SEQ ID NO: 7), and a CDR3 containing ASSWYEDETDY (SEQ ID NO: 8), along with a VHH masking portion and a vector portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing mutations in F42E and C125A, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 5, and a vector portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing mutations in F42E and C125A, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 12, and a vector portion.

[0350] In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a CDR1 containing GSIFSINVMG (SEQ ID NO: 6), a CDR2 containing AISSGGSTNYADSVKG (SEQ ID NO: 7), and a CDR3 containing ASSWYEDETDY (SEQ ID NO: 8), along with a carrier portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 5, and a carrier portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 12, and a carrier portion.

[0351] In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing CDR1 (SEQ ID NO: 6), CDR2 (SEQ ID NO: 7) containing CDR2 (SEQ ID NO: 7), and CDR3 (SEQ ID NO: 8) containing CDR3 (SEQ ID NO: 8), a carrier portion, and a targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 5, a carrier portion, and a targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 12, a carrier portion, and a targeting portion.

[0352] In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion of CDR1 containing GSIFSINVMG (SEQ ID NO: 6), a CDR2 containing AISSGGSTNYADSVKG (SEQ ID NO: 7), and a CDR3 containing ASSWYEDETDY (SEQ ID NO: 8), a vector portion, and an anti-PD1 targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 5, a vector portion, and an anti-PD1 targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 12, a vector portion, and an anti-PD1 targeting portion.

[0353] In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion of CDR1 containing GSIFSINVMG (SEQ ID NO: 6), a CDR2 containing AISSGGSTNYADSVKG (SEQ ID NO: 7), and a CDR3 containing ASSWYEDETDY (SEQ ID NO: 8), a vector portion, and an anti-PD1 targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 5, a vector portion, and an anti-PD1 targeting portion. In some embodiments, the masked cytokine comprises an attenuated IL-2 cytokine containing the amino acid sequence of SEQ ID NO: 4, a VHH masking portion containing the amino acid sequence of SEQ ID NO: 12, a vector portion, and an anti-PD1 targeting portion.

[0354] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises a targeting portion, a first Fc polypeptide, a first adaptor, and an attenuated IL-2 cytokine from the N-terminus to the C-terminus, and wherein the second polypeptide comprises a targeting portion, a second Fc polypeptide, a second adaptor, and a VHH masking portion from the N-terminus to the C-terminus.

[0355] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide, a first adapter, and an attenuated IL-2 cytokine comprising an F42E mutation, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide, a second adapter, and a VHH masking portion.

[0356] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a first Fc polypeptide, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a second Fc polypeptide, a second adapter, and a VHH masking moiety.

[0357] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a first Fc polypeptide, a first adaptor, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a second Fc polypeptide, a second adaptor, and a VHH masking moiety, and wherein the VHH producing moiety comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0358] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a first Fc polypeptide, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a second Fc polypeptide, a second adapter, and the VHH masking moiety of SEQ ID NO: 5.

[0359] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a first Fc polypeptide, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting moiety, a second Fc polypeptide, a second adapter, and the VHH masking moiety of SEQ ID NO: 12.

[0360] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and a VHH masking portion, and wherein the VHH generating portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0361] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and the VHH masking portion of SEQ ID NO: 5.

[0362] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine comprising mutations of F42E and C125A, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and the VHH masking portion of SEQ ID NO: 12.

[0363] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and a VHH masking portion, and wherein the VHH generating portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0364] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and a VHH masking portion of SEQ ID NO: 5.

[0365] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter, and a VHH masking portion of SEQ ID NO: 12.

[0366] In some implementations, the second connector is cuttable.

[0367] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking portion, and wherein the VHH generating portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0368] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking portion of SEQ ID NO: 5.

[0369] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, a targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking portion of SEQ ID NO: 12.

[0370] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting portion, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting portion, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking portion, and wherein the VHH generating portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0371] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting moiety, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting moiety, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking moiety of SEQ ID NO: 5.

[0372] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting moiety, a first Fc polypeptide comprising mutations of S354C, T366W, N297A, H435R, and Y435F, a first adapter, and an attenuated IL-2 cytokine of SEQ ID NO: 4, and wherein the second polypeptide comprises, from the N-terminus to the C-terminus, an anti-PD1 targeting moiety, a second Fc polypeptide comprising mutations of Y349C, T366S, L368A, Y407V, and N297A, a second adapter comprising a cleavage peptide, and a VHH masking moiety of SEQ ID NO: 12.

[0373] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion, and wherein the third and fourth polypeptides comprise an anti-PD1 targeting portion.

[0374] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine comprising F42E and C125A mutations, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion, and wherein the third and fourth polypeptides comprise an anti-PD1 targeting portion.

[0375] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine comprising F42E and C125A mutations, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion, wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion, and wherein the VHH masking portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0376] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine comprising F42E and C125A mutations, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and the VHH masking portion of SEQ ID NO: 5, and wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion.

[0377] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine comprising F42E and C125A mutations, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and the VHH masking portion of SEQ ID NO: 12, and wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion.

[0378] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion, wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion, and wherein the VHH masking portion comprises CDR1 of GSIFSINVMG (SEQ ID NO: 6), CDR2 of AISSGGSTNYADSVKG (SEQ ID NO: 7), and CDR3 of ASSWYEDETDY (SEQ ID NO: 8).

[0379] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion of SEQ ID NO: 5, and wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion.

[0380] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises an anti-PD1 targeting portion, a first Fc polypeptide, a linker, and an attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises an anti-PD1 targeting portion, a second Fc polypeptide, a linker, and a VHH masking portion of SEQ ID NO: 12, and wherein the third and fourth polypeptides comprise the anti-PD1 targeting portion.

[0381] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, a linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, a linker, and the VHH masking portion of SEQ ID NO: 5, and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0382] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, a linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4, wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, a linker, and the VHH masking portion of SEQ ID NO: 12, and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0383] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, an uncleavable linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4; wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, a cleavable linker, and the VHH masking portion of SEQ ID NO: 5; and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0384] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, an uncleavable linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4; wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, a cleavable linker, and the VHH masking portion of SEQ ID NO: 12; and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0385] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, an uncleavable linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4; wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, the cleavable linker of SEQ ID NO: 34, and the VHH masking portion of SEQ ID NO: 5; and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0386] In one aspect, the present invention particularly provides a masked cytokine comprising a first polypeptide, a second polypeptide, a third polypeptide, and a fourth polypeptide, wherein the first polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the first Fc polypeptide of SEQ ID NO: 21, an uncleavable linker, and the attenuated IL-2 cytokine of SEQ ID NO: 4; wherein the second polypeptide comprises the anti-PD1 targeting portion of SEQ ID NO: 170, the second Fc polypeptide of SEQ ID NO: 20, the cleavable linker of SEQ ID NO: 34, and the VHH masking portion of SEQ ID NO: 12; and wherein the third polypeptide and the fourth polypeptide each comprise the anti-PD1 targeting portion of SEQ ID NO: 176.

[0387] In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 30-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 50-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 100-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 200-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 300-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 500-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 700-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 800-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 1000-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 3000-fold compared to IL-2 cytokines without the masking portion. In some embodiments, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is greater than 5000-fold compared to IL-2 cytokines without the masking portion.

[0388] In some implementations, the masked or targeted cytokine is cleaved by at least one MMP, wherein Kcat / Km is greater than 10. 4 M -1 s -1In some implementations, the masked or targeted cytokine is cleaved by at least two MMPs, where Kcat / Km is greater than 10. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least three MMPs, where Kcat / Km is greater than 10. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least four MMPs, where Kcat / Km is greater than 10. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least five MMPs, where Kcat / Km is greater than 10. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is composed of at least one MMP, wherein Kcat / Km is greater than 5 x 10⁻⁶. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least two MMPs, where Kcat / Km is greater than 5 x 10^6. 4 M -1 s -1 In some implementations, the masked or targeted cytokines are cleaved by at least three MMPs, where Kcat / Km is greater than 5 x 10^6. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least four MMPs, where Kcat / Km is greater than 5 x 10^6. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least five MMPs, where Kcat / Km is greater than 5 x 10^5. 4 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least one MMP, wherein Kcat / Km is greater than 10. 5 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least two MMPs, where Kcat / Km is greater than 10. 5 M - 1s -1 In some implementations, the masked or targeted cytokine is cleaved by at least three MMPs, where Kcat / Km is greater than 10. 5 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least four MMPs, where Kcat / Km is greater than 10. 5 M -1 s -1 In some implementations, the masked or targeted cytokine is cleaved by at least five MMPs, where Kcat / Km is greater than 10. 5 M -1 s -1 .

[0389] In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 5-fold compared to IL-2 cytokines without the masking portion. In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 4-fold compared to IL-2 cytokines without the masking portion. In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 3-fold compared to IL-2 cytokines without the masking portion. In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 2-fold compared to IL-2 cytokines without the masking portion. In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 1-fold compared to IL-2 cytokines without the masking portion. In some embodiments, after protease cleavage, the EC50 value of the masked cytokine or targeted cytokine based on pSTAT5 activity on CD8+ T cells is within 0.8-fold compared to IL-2 cytokines without the masking portion.

[0390] In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 2%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 3%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 4%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 2%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 5%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 7%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 8%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 10%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 12%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 14%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 15%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 20%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 30%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 40%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 50%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 55%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is at least 60%. In some embodiments, the percentage of molecules cleaved from masked or targeted cytokines in the tumor is below the lower limit of quantification (LLOQ) in plasma.

[0391] In some embodiments, the masked or targeted cytokine has at least 40% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 50% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 55% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 60% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 65% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 70% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 75% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 80% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokine has at least 85% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokines have at least 90% tumor growth inhibition (TGI). In some embodiments, the masked or targeted cytokines have at least 95% tumor growth inhibition (TGI).

[0392] Masked IL-2 cytokines

[0393] In some embodiments, the masked cytokine of the present invention comprises a masking portion, a carrier portion, and a cytokine or a variant thereof. In some embodiments, the masked cytokine comprises a masking portion, an Fc domain as a carrier portion, and a cytokine or a variant thereof. In some embodiments, the masked cytokine comprises a VHH antibody as a masking portion, a carrier portion, and a cytokine or a variant thereof. In some embodiments, the masked cytokine comprises a VHH antibody as a masking portion, an Fc domain as a carrier portion, and a cytokine or a variant thereof.

[0394] In some implementations, the IL-2 peptide is linked to the Fc domain via a cleavable or non-cleavable linker.

[0395] In some embodiments, the masking portion is attached to the Fc domain via a cleavable or non-cleavable linker. In some embodiments, the targeting portion is attached to the Fc domain with or without a cleavable or non-cleavable linker. In some embodiments, the masking portion is attached to the Fc peptide via a cleavable linker. In some embodiments, the masking portion is attached to the Fc peptide via a non-cleavable linker. In some embodiments, the IL-2 peptide is attached to the Fc peptide via a cleavable linker. In some embodiments, the IL-2 peptide is attached to the Fc peptide via a non-cleavable linker.

[0396] In some embodiments, the first polypeptide comprises SEQ ID NO: 191. In some embodiments, the second polypeptide comprises SEQ ID NO: 192. In some embodiments, the third polypeptide comprises SEQ ID NO: 176. In some embodiments, the first polypeptide comprises SEQ ID NO: 191, the second polypeptide comprises SEQ ID NO: 192, and the third polypeptide comprises SEQ ID NO: 176.

[0397] In some embodiments, the masked cytokine comprises three polypeptides: (a) a first polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 191; (b) a second polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 193; and (c) a third polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 176.

[0398] In some embodiments, the first polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 191. In some embodiments, the first polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 191.

[0399] In some embodiments, the second polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 193. In some embodiments, the second polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 193.

[0400] In some embodiments, the third polypeptide comprises an amino acid sequence having at least 90% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 92% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 95% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 96% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 97% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 98% identity with SEQ ID NO: 176. In some embodiments, the third polypeptide comprises an amino acid sequence having at least 99% identity with SEQ ID NO: 176.

[0401] In some embodiments, the masked cytokine comprises three polypeptides: (a) a first polypeptide comprising the amino sequence of SEQ ID NO: 191, (b) a second polypeptide comprising the amino sequence of SEQ ID NO: 193, and (c) a third polypeptide comprising the amino sequence of SEQ ID NO: 176.

[0402] Table 13. Exemplary sequences of masked cytokines.

[0403]

[0404] Targeted IL-2 cytokines

[0405] In some implementations, the masked IL-2 cytokine also includes a targeting portion for forming a targeted IL-2 cytokine.

[0406] In some embodiments, the targeted cytokine of the present invention comprises a masking portion, a carrier portion, a targeting portion, and a cytokine or a variant thereof. In some embodiments, the targeted cytokine comprises a masking portion, an Fc domain as a carrier portion, a targeting portion, and a cytokine or a variant thereof. In some embodiments, the targeted cytokine comprises a VHH antibody as a masking portion, a carrier portion, a targeting portion, and a cytokine or a variant thereof. In some embodiments, the targeted cytokine comprises a VHH antibody as a masking portion, an Fc domain as a carrier portion, a targeting portion, and a cytokine or a variant thereof.

[0407] In some implementations, the IL-2 peptide is linked to the Fc domain via a cleavable or non-cleavable linker.

[0408] In some embodiments, the masking portion is attached to the Fc domain via a cleavable or non-cleavable linker. In some embodiments, the targeting portion is attached to the Fc domain with or without a cleavable or non-cleavable linker. In some embodiments, the masking portion is attached to the Fc peptide via a cleavable linker. In some embodiments, the masking portion is attached to the Fc peptide via a non-cleavable linker. In some embodiments, the IL-2 peptide is attached to the Fc peptide via a cleavable linker. In some embodiments, the IL-2 peptide is attached to the Fc peptide via a non-cleavable linker.

[0409] In some embodiments, the targeted cytokine comprises: a first polypeptide comprising a heavy chain of the targeting portion and a first Fc polypeptide linked to the cytokine via an uncleavable linker in the N-to-C-terminal direction; a second polypeptide comprising a heavy chain of the targeting portion and a second Fc polypeptide linked to a masking portion via a cleavable linker in the N-to-C-terminal direction; and a third polypeptide comprising a light chain of the targeting portion associated with the heavy chain of the targeting portion.

[0410] In some embodiments, the targeted cytokine comprises: a first polypeptide comprising a heavy chain of the targeting portion and a first Fc polypeptide linked to the cytokine via a cleavable linker in the N-to-C-terminal direction; a second polypeptide comprising a heavy chain of the targeting portion and a second Fc polypeptide linked to a masking portion via an uncleavable linker in the N-to-C-terminal direction; and a third polypeptide comprising a light chain of the targeting portion associated with the heavy chain of the targeting portion.

[0411] In some embodiments, the targeted cytokine comprises: a first polypeptide comprising an scFv targeting portion (e.g., a heavy chain variable region fused with a light chain variable region (VH-VL or VL-VH)) and a first Fc polypeptide linked to the cytokine via an uncleavable linker in the N-to-C-terminal direction; and a second polypeptide comprising an scFv targeting portion (e.g., a heavy chain variable region fused with a light chain variable region (VH-VL or VL-VH)) and a second Fc polypeptide linked to a masking portion via a cleavable linker in the N-to-C-terminal direction.

[0412] In some embodiments, the targeted cytokine comprises: a first polypeptide comprising an scFv targeting portion (e.g., a heavy chain variable region fused with a light chain variable region (VH-VL or VL-VH)) and a first Fc polypeptide linked to the cytokine via a cleavable linker in the N-to-C-terminal direction; and a second polypeptide comprising an scFv targeting portion (e.g., a heavy chain variable region fused with a light chain variable region (VH-VL or VL-VH)) and a second Fc polypeptide linked to a masking portion via an uncleavable linker in the N-to-C-terminal direction.

[0413] Treatment

[0414] This document provides a method for treating or preventing a disease in a subject, comprising administering to the subject an effective amount of any masking and / or targeting cytokine or a combination thereof described herein. In some embodiments, a method for treating or preventing a disease in a subject is provided, comprising administering to the subject any composition described herein. In some embodiments, the subject (e.g., a human patient) has been diagnosed with cancer or is at risk of developing such a condition. In some embodiments, a method for treating or preventing a disease in a subject is provided, comprising administering to the subject an effective amount of any targeting cytokine or a combination thereof described herein, wherein the targeting cytokine is activated upon enzymatic cleavage. In some embodiments, the targeting cytokine is activated in a tumor microenvironment. The targeting cytokine has therapeutic activity after cleavage. Therefore, in some embodiments, the active agent is a cleavage product. In some embodiments, the targeted masked cytokine has therapeutic activity after the masked portion has been cleaved. Therefore, in some embodiments, the active agent is an unmasked targeting cytokine.

[0415] In some embodiments, masked cytokines are activated within the tumor microenvironment. The masked cytokines possess therapeutic activity after the masked portion has been cleaved. Therefore, in some embodiments, the active agent is an unmasked cytokine. In some embodiments, the masked cytokines possess therapeutic activity after cleavage. Therefore, in some embodiments, the active agent is a cleavage product.

[0416] In one aspect, the present invention provides a method for treating a condition or disease, such as cancer, using any of the masked cytokines or compositions described herein. Conditions or diseases treatable with the formulations of the present invention include leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma) or testicular cancer.

[0417] In some embodiments, this document provides a method for treating or preventing cancer by administering any masked cytokine or composition described herein. In some embodiments, this document provides a method for treating or preventing cancer by administering any masked cytokine or composition described herein in combination with an anticancer agent. The anticancer agent can be any agent capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell survival. In some implementation schemes, the anticancer agent is selected from the group consisting of: PD-1 inhibitors, EGFR inhibitors, HER2 inhibitors, VEGFR inhibitors, CTLA-4 inhibitors, BTLA inhibitors, B7H4 inhibitors, B7H3 inhibitors, CSFIR inhibitors, HVEM inhibitors, CD27 inhibitors, KIR inhibitors, NKG2A inhibitors, NKG2D agonists, TWEAK inhibitors, ALK inhibitors, CD52-targeting antibodies, CCR4-targeting antibodies, PD-L1 inhibitors, KIT inhibitors, PDGFR inhibitors, BAFF inhibitors, HDAC inhibitors, VEGF ligand inhibitors, CD19-targeting molecules, FOFR1-targeting molecules, DFF3-targeting molecules, DKK1-targeting molecules, MUC1-targeting molecules, MUG 16-targeting molecules, PSMA-targeting molecules, MSFN-targeting molecules, NY-ESO-1-targeting molecules, B7H3-targeting molecules, B7H4-targeting molecules, BCMA-targeting molecules, CD29-targeting molecules, CD151-targeting molecules, CD 123-targeting molecules, CD33-targeting molecules, CD37-targeting molecules, CDH19-targeting molecules, CEA-targeting molecules, and sealing protein 18.Targeted molecules, including CFEC12A, EGFRVIII, EPCAM, EPHA2, FCRH5, FLT3, GD2, phosphatidylinositol proteoglycan 3, gpA33, GPRC5D, IL-23R, IL-1RAP, MCSP, RON, ROR1, STEAP2, TfR, CD166, TPBG, TROP2, proteasome inhibitors, ABE inhibitors, CD30 inhibitors, FLT3 inhibitors, MET inhibitors, RET inhibitors, IL-1(3) inhibitors, MEK inhibitors, ROS1 inhibitors, BRAE inhibitors, CD38 inhibitors, and RANKE inhibitors. The list includes inhibitors such as B4GALNT1 inhibitors, SLAMF7 inhibitors, IDH2 inhibitors, mTOR inhibitors, CD20-targeting antibodies, BTK inhibitors, PI3K inhibitors, FLT3 inhibitors, PARP inhibitors, CDK4 inhibitors, CDK6 inhibitors, EGFR inhibitors, RAF inhibitors, JAK1 inhibitors, JAK2 inhibitors, JAK3 inhibitors, IL-6 inhibitors, IL-17 inhibitors, Smoothened inhibitors, IL-6R inhibitors, BCL2 inhibitors, PTCH inhibitors, PIGF inhibitors, TGFB inhibitors, CD28 agonists, CD3 agonists, CD40 agonists, GITR agonists, OX40 agonists, VISTA agonists, CD137 agonists, LAG3 inhibitors, TIM3 inhibitors, TIGIT inhibitors, and IL-2R inhibitors.

[0418] In some implementations, this document provides a method for treating or preventing cancer by administering any masked cytokines described herein in combination with an anti-inflammatory agent. The anti-inflammatory agent can be any agent capable of preventing, counteracting, inhibiting, or otherwise reducing inflammation.

[0419] In some embodiments, the anti-inflammatory agent is a cyclooxygenase (COX) inhibitor. The COX inhibitor can be any agent that inhibits COX-1 and / or COX-2 activity. In some embodiments, the COX inhibitor selectively inhibits COX-1 (i.e., the COX inhibitor inhibits COX-1 activity more than its COX-2 activity). In some embodiments, the COX inhibitor selectively inhibits COX-2 (i.e., the COX inhibitor inhibits COX-2 activity more than its COX-1 activity). In some embodiments, the COX inhibitor inhibits both COX-1 and COX-2.

[0420] In some implementations, the COX inhibitor is a selective COX-1 inhibitor and is selected from the group consisting of: SC-560, FR122047, P6, mofezolac, TFAP, flurbiprofen, and ketoprofen. In some implementations, the COX inhibitor is a selective COX-2 inhibitor and is selected from the group consisting of: celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, valdecoxib, etoricoxib, benzopyran derivatives, benzodihydropyran derivatives, N-(2-cyclohexyloxynitrobenzyl)methanesulfonamide, parecoxib, lumiracoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimesulide, flosulide, NS-398, L- 745337, RWJ-63556, L-784512, darbufelone, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, and SD-8381. In some embodiments, the COX inhibitor is selected from the group consisting of ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tolmetin, piroxicam, and meclofenamate.In some implementation schemes, the COX inhibitor is selected from the group consisting of: SC-560, FR122047, P6, monoxazol, TFAP, flurbiprofen, ketoprofen, celecoxib, rofecoxib, meloxicam, piroxicam, deracoxib, parecoxib, vardicoxib, etoricoxib, benzopyran derivatives, benzodihydropyran derivatives, N-(2-cyclohexyloxynitrophenyl)methanesulfonamide, parecoxib, romecoxib, RS 57067, T-614, BMS-347070, JTE-522, S-2474, SVT-2016, CT-3, ABT-963, SC-58125, nimesulide, fluoxuclizine, NS-398, L- 745337, RWJ-63556, L-784512, dabufone, CS-502, LAS-34475, LAS-34555, S-33516, diclofenac, mefenamic acid, SD-8381, ibuprofen, naproxen, ketorolac, indomethacin, aspirin, naproxen, tometidine, piroxicam, and mefenamic acid salt.

[0421] In some embodiments, the anti-inflammatory agent is an NF-κB inhibitor. An NF-κB inhibitor can be any agent that inhibits the activity of the NF-κB pathway. In some embodiments, the NF-κB inhibitor is selected from the group consisting of: IKK complex inhibitors, IκB degradation inhibitors, NF-κB nuclear translocation inhibitors, p65 acetylation inhibitors, NF-κB DNA binding inhibitors, NF-κB transactivation inhibitors, and p53 induction inhibitors.

[0422] In some embodiments, the IKK complex inhibitor is selected from the group consisting of: TPCA-1, NF-KB activation inhibitor VI (BOT-64), BMS-345541, ammexanox, SC-514 (GK-01140), IMD-0354, and IKK-16. In some embodiments, the IKB degradation inhibitor is selected from the group consisting of: BAY-11-7082, MG-115, MG-132, lactacystin, epoxomicin, parthenolide, carfilzomib, and MLN-4924 (pevonedistat). In some embodiments, the NF-KB nuclear translocation inhibitor is selected from the group consisting of JSH-23 and rolipram. In some embodiments, the p65 acetylation inhibitor is selected from the group consisting of gallic acid and astaxanthin. In some embodiments, the NF-KB DNA binding inhibitor is selected from the group consisting of GYY-4137, p-XSC, CV-3988, and prostaglandin E2 (PGE2). In some embodiments, the NF-KB transactivation inhibitor is selected from the group consisting of LY-294002, wortmannin, and mesalamine. In some embodiments, the p53 induction inhibitor is selected from the group consisting of quinacrine and flavopiridol. In some implementations, the NF-KB inhibitor is selected from the group consisting of: TPCA-1, NF-KB activation inhibitor VI (BOT-64), BMS-345541, amoxicillin, SC-514 (GK-01140), IMD-0354, IKK-16, BAY-11-7082, MG-115, MG-132, lactocinol, cyclooxygenin, parthenolide, carfilzomib, MLN-4924 (penostat), JSH-23, cyclophosphamide, gallic acid, ascorbic acid, GYY-4137, p-XSC, CV-3988, prostaglandin E2 (PGE2), LY-294002, wollamin, mesalazine, quinacrine, and frapinol.

[0423] In some embodiments, this document provides a method for treating or preventing cancer by administering any masked cytokine or composition described herein in combination with an anticancer therapeutic protein. The anticancer therapeutic protein can be any therapeutic protein capable of reducing cancer growth, interfering with cancer cell replication, directly or indirectly killing cancer cells, reducing metastasis, reducing tumor blood supply, or reducing cell survival. Exemplary anticancer therapeutic proteins can be antibodies or fragments thereof, antibody derivatives, bispecific antibodies, chimeric antigen receptor (CAR) T cells, fusion proteins, or bispecific T cell adaptors (BiTE). In some embodiments, this document provides a method for treating or preventing cancer by administering any masked cytokine or composition described herein in combination with CAR-NK (natural killer) cells.

[0424] Methods for generating masked cytokine constructs

[0425] Some embodiments of the methods and compositions provided herein relate to methods for generating targeted, masked agents, such as targeted, masked cytokines. Targeted, masked cytokines as described herein can be generated and manufactured using any techniques known in the art. Targeted, masked cytokines and / or constructs can be generated using any mammalian expression system.

[0426] Some embodiments of the methods and compositions provided herein relate to methods for generating masked agents, such as masked cytokines. Masked cytokines as described herein can be generated and manufactured using any techniques known in the art. Masked cytokines and / or constructs can be generated using any mammalian expression system.

[0427] Mammalian cells are commonly used to produce recombinant proteins. These cells are derived from humans, rats, mice, and other mammals. Commonly used mammalian cell lines include, but are not limited to, HEK cells, CHO cells, recombinant CHO cells, BHK cells, NSO cells, SP2 / O-Ag14 cells, HT-1080 cells, PER.C6 cells, CAP (CEVEC's Amniocyte Production), HeLa cells, and HuH-7 (human hepatocellular carcinoma) cells. These cell lines can be used for transient gene expression or as stable cell lines for stable expression.

[0428] In some implementations, the cells are Chinese hamster ovary (CHO) cells. CHO cells can be recombinant CHO cell lines, such as CHO-K1, CHO DUX, and CHO DG44.

[0429] In some embodiments, the cells are human cells. In some embodiments, the cells are human embryonic kidney (HEK) cells or variants thereof (e.g., HKB11).

[0430] Transform host cells to express nucleic acids or vectors encoding the masked cytokines described herein. Where appropriate, engineered host cells can be cultured in conventional nutrient media.

[0431] In other implementations, non-mammalian expression systems, such as baculovirus expression systems, bacterial systems, yeast cells, insect cell lines, and plant cells, can be used to produce targeted, masked cytokines. Insect cell lines include, but are not limited to, Sf9, Sf21, and BTI 5B1-4. *E. coli* cells are commonly used bacteria for expressing recombinant proteins.

[0432] In some implementations, a cell-free protein expression system can be used to generate masked cytokines and / or constructs. The cell-free expression system can use mRNA transcribed from a DNA construct containing a promoter operatively linked to a nucleic acid encoding a polypeptide or a fragment thereof.

[0433] Example

[0434] Although certain compounds, compositions and methods of the present invention have been specifically described according to some embodiments, the following examples are for illustrative purposes only and are not intended to limit the scope of these compounds.

[0435] Example 1. Effective masking and activation of masked cytokines

[0436] This embodiment demonstrates that the masked IL-2 cytokine of the present invention is effectively masked by the VHH masking portion, and is activated once the VHH masking portion is released by tumor-specific proteases. In some embodiments, the masked cytokine comprises Figure 1A The illustrated form. In some implementations, the masked cytokines contain... Figure 1B the form exemplified.

[0437] Table 7. Exemplary masked IL-2 cytokines

[0438]

[0439] In this specific embodiment, masked IL-2 cytokines as shown in Table 7 were prepared, wherein the purity was greater than at least 97%. All the masked IL-2 cytokines tested were able to inhibit PD-1 / PD-L1 interaction and were effectively cleaved by at least one MMP (data not shown).

[0440] pSTAT5 activity was measured on human PBMCs. Briefly, hPBMCs were activated with anti-CD3 and anti-CD28. Masked cytokines (whether cleaved or uncleaved with MMPs) were titrated onto hPBMCs. Unmasked controls and recombinant human IL-2 (rhIL-2) were used as controls. hPBMCs were stained against CD4+ T cells, CD8+ T cells, and pSTAT5. A high EC50 value for pSTAT50 activity on CD8+ T cells indicated effective cytokine masking. An EC50 value greater than 300-fold for masked (uncleaved) cytokines indicated effective IL-2 cytokine masking compared to the unmasked control.

[0441] Table 8. pSTAT on hPBMC

[0442]

[0443] Figure 2 Table 1 shows the percentage of pSTAT in CD8+ T cells with different concentrations of rhIL-2, the unmasked control, and C5 and C6. Table 8 shows the mean EC50 values ​​of the masked constructs for pSTAT activity in CD8+ T cells. Table 8 shows that C5 and C6 both had EC50 values ​​greater than approximately 7400-fold and approximately 8500-fold, respectively, compared to the unmasked control, indicating that the cytokines were effectively masked. Furthermore, when the masked portion was cleaved by MMPs, the IL-2 cytokine in the C5 and C6 constructs regained its activity, as shown by the low fold change values ​​in Table 8 compared to the unmasked control.

[0444] Example 2. Selective tumor resection in vitro using masked IL-2 cytokines

[0445] This embodiment confirms that the masked IL-2 cytokine of the present invention is activated by human tumors, but not by human plasma.

[0446] Tumor and plasma samples from different cancer indications—melanoma, head and neck cancer (HNC), lung cancer, colon cancer, and renal cell carcinoma (RCC)—were cultured and treated using the constructs shown in Table 9. The percentage of cleaved molecules was determined 24 hours post-treatment. Preferably, the percentage of cleaved molecules in the tumor was greater than 5% in at least one tested cancer indication, and the percentage of cleaved molecules was below the lower limit of quantitation (LLOQ) in plasma.

[0447] Table 9. Exemplary masked IL-2 cytokines

[0448]

[0449] like Figure 3AAs shown, the C5 and C6 constructs were cleaved at a significantly higher rate than the uncleavable control, demonstrating activation of C5 and C6 in multiple oncology indications. Furthermore, minimal cleavage of masked cytokines was observed in human plasma across all indications. Figure 3B ).

[0450] Example 3. In vivo efficacy of masked IL-2 cytokines

[0451] This embodiment demonstrates that the masked IL-2 cytokine of the present invention is preferentially activated by tumor-specific proteases in tumors and reduces tumor volume in vivo. Figure 1A As shown in Table 10, the masked cytokines tested contained three polypeptide chains (one copy of chain A, one copy of chain B, and two copies of chain C).

[0452] Table 10. Masked IL-2 cytokines

[0453]

[0454] Masked IL-2 cytokines are tumor-selective.

[0455] To determine whether the masked IL-2 cytokine of this invention is preferentially activated in tumors, 0.5 x 10⁻⁶ ppm was subcutaneously injected into mice with hPD-1 (n=5 in each group). 6 MC38 tumor cells. When the tumor volume reaches approximately 350-400 mm. 3 Treatment was initiated at that time. On days 0 and 3, mice received constructs C5 and C6, unmasked controls, or mediators. On day 6, mice were sacrificed, and blood, spleen, and tumor tissue were analyzed using FACS. Figure 4 The profiles of CD8 T cells in spleen cells and the tumor microenvironment are shown, illustrating that the masked cytokines C5 and C6 confirm the tissue-selective expansion of CD8 T cells within the tumor.

[0456] The activity of IL-2 cytokines was also measured in tumors, plasma, and spleen. Figure 5 The results showed that no detectable percentage of active molecules were observed in plasma and spleen for the C5 and C6 treatment groups. Active IL-2 cytokines were detected in tumors from all C5 and C6 treatment groups, confirming that C5 and C6 are tumor-selective and have minimal activation in plasma or spleen.

[0457] Masked IL-2 cytokines are effective in vivo.

[0458] To determine the in vivo efficacy of the masked IL-2 cytokine of the present invention, 0.5 x 10⁻⁶ mmol / L was subcutaneously injected into the right side of hPD-1-containing mice (n=8 in each group). 6MC38 tumor cells. When the tumor volume reaches approximately 100-150 mm. 3 Treatment was initiated at that time. Mice were administered constructs C5, C6, unmasked controls, or mediators on days 0 and 5. Tumor volume was measured intermittently until day 12. Figure 6 The results showed that, compared with the control group, both C5 and C6 significantly inhibited tumor growth. The tumor growth inhibition rates of C5 and C6 were 66% and 64%, respectively.

[0459] Next, 1 x 10⁻⁶ mmol / L was subcutaneously injected into the right side of mice with hPD-1 (n=8 in each group). 6 MB49 tumor cells. On days 0 and 5, mice were administered the C6 construct shown in Table 11, control-masked IL-2 cytokines, control-masked cytokines plus anti-PD1 antibody, anti-PD1 antibody, or mediators. The control-masked IL-2 cytokines used in this study comprised two peptides: (1) [Fc domain]-[uncleavable linker]-[non-α IL-2]; and (2) [Fc domain]-[cleavable linker]-[IL-2 receptor β-masking moiety];

[0460] Table 11. Treatment groups in in vivo studies using MB49 tumor mice

[0461]

[0462] like Figure 7A As shown, the masked IL-2 cytokine of this invention inhibits tumor growth, demonstrating significant single-therapy activity. Furthermore, overall survival was significantly higher than any other group tested ( Figure 7B ).

[0463] Example 4. Exemplary masked IL-2 cytokine blocks PD-1 / PD-L1 interaction

[0464] This embodiment demonstrates the ability of the exemplary masked IL-2 cytokine to maintain PD-1 targeting and block the PD-1 / PD-L1 axis.

[0465] Test exemplary masked IL-2 cytokines and a reference anti-PD1 antibody with PD-1 blocking activity using the Promega PD-1 / PD-L1 blocking bioassay. Set up and run the bioassay according to the manufacturer's protocol.

[0466] In short, the day before the assay, prepare the cell recovery medium provided with the bioassay kit by adding the provided FBS (90% Ham's F-12 / 10% FBS). Gently thaw one vial of PD-L1aAPC / CHO-K1 cells in a 37°C water bath for 3 minutes. Add the thawed vial of PD-L1aAPC / CHO-K1 cells 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 prepared with tissue culture. Cap the plate and incubate overnight (37°C, 5% CO2).

[0467] On the second day, the assay plate containing PD-L1 aAPC / CHO-K1 cells was removed from the incubator, and 95 µL of recovery medium was removed from each well. Serial dilutions of the test sample, exemplary masked IL-2 cytokine, and reference antibody were prepared in the provided assay buffer (99% RPMI 1640 / 1% FBS). 40 µL aliquots of the serial dilutions were added to the wells containing PD-L1 aAPC / CHO-K1 cells, where the final concentration of the test sample ranged from 0.033 to 50 nM. Next, PD-1 effector cells were prepared by thawing a vial of these cells in a 37°C water bath for 3 minutes and adding the thawed effector cells to the pre-warmed assay buffer. The cell suspension was mixed by inverting the plate twice before adding 40 µL of effector cell suspension to each well. The assay plate was capped and incubated for 6 hours (37°C, 5% CO2).

[0468] After 6 hours of incubation, remove the assay plate from the incubator and allow it to equilibrate to 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 luminescence.

[0469] In this assay, an exemplary masked 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 the interaction, the exemplary masked IL-2 cytokine released an inhibitory signal, leading to increased T cell receptor (TCR) signaling and luminescence. Figure 8 The figure shows the fold induction of luminescence in an exemplary masked IL-2 cytokine.

[0470] Data analysis was performed in GraphPad Prism. Induction fold was calculated by subtracting background luminescence from the luminescence of the test sample, then dividing by the result of subtracting background luminescence from the antibody-free control (RLU [antibody-background] / RLU [antibody-free control-background]). The calculated EC50 for each exemplary masked IL-2 cytokine was calculated using a nonlinear regression of [agonist] relative to the response with a variable slope (four parameters). 50 Values ​​(as shown in Table 12).

[0471] Table 12. EC50 of exemplary masked IL-2 cytokines 50 value

[0472]

[0473] 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.

[0474] Equivalent solution

[0475] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experiments. The scope of the invention is not intended to be limited to the foregoing description, but rather as set forth in the following claims:

Claims

1. A masked cytokine comprising: a) Attenuated interleukin-2 (IL-2) peptide, b) The masking portion, which contains only heavy chain antibodies (VHH). c) The carrier portion, and d) Anti-PD1 targeting component.

2. The masked cytokine of claim 1, wherein the attenuated IL-2 polypeptide contains mutations at amino acid positions R38, F42, K43, Y45, E62, L72 and / or C125 compared to the sequence of mature IL-2 having SEQ ID NO:

2.

3. The masked cytokine of claim 2, wherein the mutation is R38G, R38A, F42A, F42K, F42E, K43A, Y45A, Y45N, Y45R, E62R, E62S, E62A, L72G, C125A and / or C125S.

4. The masked cytokine of any one of claims 2 or 3, wherein the attenuated IL-2 polypeptide comprises the mutation: a) C125A; b) F42E, C125A; c) E62S, C125A; d) F42A, C125A; e) Y45R, C125A; f) F42S, E62S, C125A; g) F42K; h) F42A, Y45A, L72G, C125A; i) E62R; j) Y45N; k) K43A; l) Y45A, E62S; or m) R38G, Y45A, E62S.

5. The masked cytokine of claim 4, wherein the attenuated IL-2 polypeptide comprises F42E and C125A.

6. The masked cytokine of claim 5, wherein the attenuated IL-2 polypeptide comprises SEQ ID NO:

4.

7. The masked cytokine as described in any of the preceding claims, wherein the VHH specifically binds to the IL-2 polypeptide.

8. The masked cytokine as claimed in any of the preceding claims, wherein the VHH comprises HCDR1 containing the amino acid sequence GSIFSINVMG (SEQ ID NO: 6), HCDR2 containing the amino acid sequence AISSGGSTNYADSVKG (SEQ ID NO: 7), and HCDR3 contains an amino acid sequence selected from ASSWYEDETDY (SEQ ID NO: 8).

9. The masked cytokine as described in any of the preceding claims, wherein the VHH comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

5.

10. The masked cytokine as described in any of the preceding claims, wherein the VHH comprises the amino acid sequence of SEQ ID NO:

5.

11. The masked cytokine as described in any of the preceding claims, wherein the VHH further comprises a C-terminal amino acid extension.

12. The masked cytokine of claim 11, wherein the C-terminal extension comprises 1-5 amino acids.

13. The masked cytokine of claim 11, wherein the C-terminal extension is selected from the group consisting of A, AS, AST, AAA, AH, GS, PP, PPP and GP.

14. The masked cytokine of claim 13, wherein the masking portion comprises a C-terminal amino acid extension containing AAA.

15. The masked cytokine of claim 14, comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

12.

16. The masked cytokine of claim 15, comprising the amino acid sequence of SEQ ID NO:

12.

17. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises an antigen-binding domain selected from the group consisting of: Fab, single-chain Fv (scFv), single-domain antibody (VHH), one or more CDRs, variable heavy chain (VH), variable light chain (VL), Fab-like bispecific antibody (bsFab), single-domain antibody-linked Fab (s-Fab), antibody, and combinations thereof.

18. 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: 172), The heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 173), and The heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 174).

19. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises The light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 177), The light chain CDR2 sequence of LAS (SEQ ID NO: 178), and The light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 179).

20. 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: 172), The heavy chain CDR2 sequence of INPSNGGT (SEQ ID NO: 173), The heavy chain CDR3 sequence of ARRDYRFDMGFDY (SEQ ID NO: 174), The light chain CDR1 sequence of KGVSTSGYSY (SEQ ID NO: 177), The light chain CDR2 sequence of LAS (SEQ ID NO: 178), and The light chain CDR3 sequence of QHSRDLPLT (SEQ ID NO: 179).

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

169.

22. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises a light chain variable region having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

175.

23. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises The heavy chain variable region having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 169 and The light chain variable region having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:

175.

24. The masked cytokine as claimed in any of the preceding claims, wherein the targeting portion comprises Contains the heavy chain variable region of SEQ ID NO: 169 and Contains the light chain variable region of SEQ ID NO:

175.

25. The masked cytokine as claimed in any of the preceding claims, wherein the carrier portion is a PEG molecule, albumin, albumin fragment, antibody Fc domain, or antibody or its antigen-binding fragment.

26. The masked cytokine of claim 25, wherein the carrier portion comprises an Fc domain.

27. The masked cytokine of claim 26, wherein the Fc domain comprises a first Fc polypeptide and a second Fc polypeptide.

28. The masked cytokine of claim 27, wherein the first Fc polypeptide is linked to the IL-2 polypeptide via a first linker, and the second Fc polypeptide is linked to the masking portion via a second linker, and The first or second connector contains a tumor-associated protease cleavage site, which allows the masking portion to be released from the IL-2 peptide.

29. The masked cytokine of claim 28, wherein the first connector comprises a tumor-associated protease cleavage site.

30. The masked cytokine of claim 28, wherein the second connector comprises a tumor-associated protease cleavage site.

31. The masked cytokine as claimed in any one of claims 27 to 30, wherein... i) The first Fc polypeptide contains Y349C, T366S, L368A, and Y407V mutations, and the second Fc polypeptide contains S354C and T366W mutations; or ii) The second Fc polypeptide contains Y349C, T366S, L368A and Y407V mutations, and the first Fc polypeptide contains S354C and T366W mutations.

32. The masked cytokine as claimed in any one of claims 27 to 31, wherein... i) The first Fc polypeptide contains a CH3 domain modified to weaken or eliminate binding to protein A, and ii) The second Fc polypeptide contains a CH3 domain that binds to protein A.

33. The masked cytokine of claim 32, wherein the modifications that weaken or eliminate binding to protein A according to Kabat numbering are H435R and Y436F.

34. The masked cytokine of any one of claims 27 to 33, wherein the first Fc polypeptide comprises an N297A mutation.

35. The masked cytokine of any one of claims 27 to 33, wherein the second Fc polypeptide comprises an N297A mutation.

36. The masked cytokine of any one of claims 27 to 34, wherein the first Fc polypeptide comprises an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof.

37. The masked cytokine of any one of claims 27 to 35, wherein the second Fc polypeptide comprises an IgG1, IgG2, or IgG4 Fc domain or a fragment thereof.

38. The masked cytokine of any one of claims 28 to 37, wherein the tumor-associated protease cleavage site comprises a sequence selected from Table 3.

39. The masked cytokine of any one of claims 28 to 38, wherein the tumor-associated protease cleavage site comprises MPYDLYHP (SEQ ID NO: 34).

40. A masked cytokine comprising: a) An attenuated interleukin-2 (IL-2) polypeptide containing amino acid substitutions for F42E and C125A; b) The VHH masking portion, which includes CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7 and CDR3 of SEQ ID NO: 8; c) An Fc domain comprising a first Fc polypeptide linked to the attenuated IL-2 polypeptide via a first linker and a second Fc polypeptide linked to the VHH masking portion via a second linker; and d) Anti-PD1 targeting component.

41. A masked cytokine comprising: a) An attenuated interleukin-2 (IL-2) polypeptide comprising SEQ ID NO: 4; b) The VHH masking portion, which includes SEQ ID NO: 5 or SEQ ID NO: 12; c) An Fc domain comprising a first Fc polypeptide of SEQ ID NO: 21 linked to the attenuated IL-2 polypeptide via a first connector and a second Fc polypeptide of SEQ ID NO: 20 linked to the VHH masking portion via a second connector; and d) Anti-PD1 targeting portion, which includes SEQ IDs: 169 and 175.

42. A masked cytokine comprising three polypeptides: a) The first polypeptide, which comprises a variable heavy chain with an anti-PD1 targeting moiety, a first Fc polypeptide, an uncleavable linker, and an attenuated IL-2 polypeptide. b) A second polypeptide comprising a variable heavy chain of an anti-PD1 targeting moiety, a second Fc polypeptide, a cleavable linker, and a VHH masking moiety; and c) A third polypeptide containing a variable light chain with an anti-PD1 targeting moiety.

43. The masked cytokine of claim 42, wherein the attenuated IL-2 peptide comprises F42E and C125A mutations.

44. The masked cytokine of claim 43, wherein the attenuated IL-2 polypeptide comprises SEQ ID NO:

4.

45. The masked cytokine of any one of claims 42 to 44, wherein the VHH masking portion comprises CDR1 of SEQ ID NO: 6, CDR2 of SEQ ID NO: 7, and CDR3 of SEQ ID NO:

8.

46. ​​The masked cytokine of claim 45, wherein the VHH masking portion comprises SEQ ID NO: 5 or SEQ ID NO:

12.

47. The masked cytokine of any one of claims 42 to 46, wherein the first Fc polypeptide comprises the S354C, T366W, N297A, H435R, and Y435F mutations, and the second Fc polypeptide comprises the Y349C, T366S, L368A, Y407V, and N297A mutations.

48. The masked cytokine of claim 47, wherein the first Fc polypeptide comprises SEQ ID NO: 21, and the second Fc polypeptide comprises SEQ ID NO:

20.

49. The masked cytokine of any one of claims 42 to 47, wherein the variable heavy chain of the anti-PD-1 targeting portion comprises hCDR1 of SEQ ID NO: 172, hCDR2 of SEQ ID NO: 173, and hCDR3 of SEQ ID NO: 174, and the variable light chain of the anti-PD-1 targeting portion comprises lCDR1 of SEQ ID NO: 177, lCDR2 of SEQ ID NO: 178, and lCDR3 of SEQ ID NO:

179.

50. The masked cytokine of claim 49, wherein the variable heavy chain of the anti-PD-1 targeting portion comprises SEQ ID NO: 169, and the variable light chain of the anti-PD-1 targeting portion comprises SEQ ID NO:

175.

51. The masked cytokine of any one of claims 42 to 50, wherein the cleavable adapter comprises MPYDLYHP (SEQ ID NO: 34) or SPGGGGPMPYDLYHPSGGG (SEQ ID NO: 144).

52. The masked cytokine of any one of claims 42 to 51, wherein the uncuttable linker comprises GGSSPPGGGSSGGGSGP (SEQ ID NO: 163).

53. A masked cytokine comprising three polypeptides: a) A first polypeptide, comprising: A variable heavy chain containing the anti-PD1 targeting moiety of SEQ ID NO:

169. The first Fc polypeptide of SEQ ID NO: 21 Uncuttable joints, and Attenuated IL-2 peptide of SEQ ID NO: 4; b) A second polypeptide, comprising: A variable heavy chain containing the anti-PD1 targeting moiety of SEQ ID NO:

169. The second Fc polypeptide of SEQ ID NO: 20 A cuttable connector containing MPYDLYHP (SEQ ID NO: 34), and The VHH masking portion of SEQ ID NO: 5 or SEQ ID NO: 12, and c) A third polypeptide comprising a variable light chain containing the anti-PD1 targeting moiety of SEQ ID NO:

175.

54. A masked cytokine comprising three polypeptides: a) A first polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 191; b) A second polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 192; and c) A third polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

176.

55. The masked cytokine of claim 54, wherein the first polypeptide comprises SEQ ID NO: 191, the second polypeptide comprises SEQ ID NO: 192, and the third polypeptide comprises SEQ ID NO:

176.

56. A masked cytokine comprising three polypeptides: a) A first polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 191; b) A second polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 193; and c) A third polypeptide comprising an amino sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO:

176.

57. The masked cytokine of claim 56, wherein the first polypeptide comprises SEQ ID NO: 191, the second polypeptide comprises SEQ ID NO: 193, and the third polypeptide comprises SEQ ID NO:

176.

58. The masked cytokine as claimed in any of the preceding claims, wherein, in a CD8 T cell pSTAT5 activity assay, the EC50 of the masked cytokine is greater than that of the unmasked cytokine.

59. The masked cytokine as claimed in any of the preceding claims, wherein the masked cytokine has a concentration of greater than or equal to 10 in the presence of MMP. 5 M -1 s -1 The external cutting efficiency Kcat / Km.

60. The masked cytokine as claimed in any of the preceding claims, wherein at least 5% of the masked cytokine is cleaved upon contact with a tumor-specific protease.

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

62. A vector comprising the nucleic acid as described in claim 61.

63. A host cell comprising the nucleic acid as described in claim 61.

64. A method for producing a masked cytokine as described in any one of claims 1 to 60, comprising culturing a host cell as described in claim 63 under conditions for producing the masked cytokine.

65. A pharmaceutical composition comprising a masked cytokine and a pharmaceutically acceptable carrier as described in any one of claims 1 to 60.

66. A kit comprising a masked cytokine as described in any one of claims 1 to 60 or a pharmaceutical composition as described in claim 65.

67. A method for treating or preventing a neoplastic disease in a subject, the method comprising administering to the subject an effective amount of a masked cytokine as described in any one of claims 1 to 60 or a pharmaceutical composition as described in claim 65.

68. 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 a masked cytokine as described in any one of claims 1 to 60 or a pharmaceutical composition as described in claim 65.

69. The masked cytokine as described in any one of claims 1 to 60, for use in medicine.

70. The masked cytokine as described in any one of claims 1 to 60, used in the treatment of neoplastic diseases.

71. The masked cytokine as described in any one of claims 1 to 60, used for the treatment or prevention of inflammatory diseases.

72. The masked cytokine as described in any one of claims 1 to 60, used in the treatment or prevention of autoimmune diseases.

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