Trispecific compositions comprising il-18, VEGF binding domains, and PD-1 binding domains

Abstract

The present disclosure relates anti-PD-1 / anti-VEGF immunocytokines, particularly with IL-18. Also described herein are new formats of bispecific anti-PD-1 and anti-VEGF constructs. Further described herein are IL-18 / anti-VEGF immunocytokines. Also provided herein are pharmaceutical composition and methods of treatment using the same.

Description

WSGR Docket No. 56146-747.601TRISPECIFIC COMPOSITIONS COMPRISING IL-18, VEGF BINDING DOMAINS, AND PD-1 BINDING DOMAINSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 708,087 filed October 16, 2024, and U.S. Provisional Application No. 63 / 739,331 filed December 27, 2024, which applications are incorporated herein by reference in their entirety.BRIEF SUMMARY

[0002] Described herein are multifunctional immunocytokine compositions which comprise a PD-1 binding domain, a VEGF A binding domain (also referred to herein as a “VEGF binding domain” or by similar terminology), and a cytokine. In some embodiments, the cytokine is an IL- 18 polypeptide.

[0003] In another aspect, the instant disclosure further provides an immunocytokine comprising an anti-VEGFA portion and an IL- 18 polypeptide.

[0004] In some embodiments of a multifunctional immunocytokine composition, the VEGFA binding domain and PD-1 binding domain and / or IL-18 polypeptide exhibit cooperative behavior such that binding of the VEGFA binding domain to VEGFA (e.g., VEGF dimers) results in enhanced anti-PD-1 activity and / or enhanced IL-18 activity. In some embodiments, this cooperative behavior results from a non-covalent multimerization of the immunocytokine composition when the VEGFA binding domain binds to VEGFA. For example, in some embodiments, a first molecule of the multifunctional immunocytokine can bind to a monomer of a VEGF dimer in situ and a second molecule of the multifunctional immunocytokine can bind to the other monomer of the VEGF dimer, thus forming, in essence, a non-covalent multimer of the multifunctional immunocytokine. Once formed, the multimer can exhibit enhanced anti-PD-1 and / or enhanced IL-18 activity (e.g., by avidity -like effects) compared to the multifunctional immunocytokine without the presence of VEGFA. Thus, in some embodiments, multifunctional immunocytokines of the instant disclosure can provide for delivery of PD-1 and / or IL-18 to a subject with increased therapeutic index by optimizing the activity in tumor microenvironments where VEGFA is present.

[0005] In some embodiments, a multifunctional immunocytokine of the instant disclosure utilizes an IL- 18 polypeptide which retains a certain threshold activity when incorporated into an immunocytokine composition. In some embodiments, the retention of this threshold activity allows for a suitably therapeutically effective molecule.WSGR Docket No. 56146-747.601

[0006] Further provided herein are methods of treating cancer and other disease with the aforementioned composition, as well as pharmaceutical compositions comprising the same.

[0007] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0008] In an aspect, the present disclosure provides a multifunctional immunocytokine composition, comprising: a) a first binding domain targeting programmed cell death protein 1 (PD-1); b) a second binding domain targeting vascular endothelial growth factor A (VEGFA); and c) a cytokine, wherein each of the first binding domain, the second binding domain, and the cytokine are in covalent association. In some embodiments, the cytokine is selected from an interleukin, a TNF family cytokine, an interferon, a TGF-b family cytokine, and a chemokine.

[0009] In some embodiments, the cytokine is an IL-18 polypeptide. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to the sequence set forth in SEQ ID NO: 701. In some embodiments, the IL-18 polypeptide exhibits reduced binding to IL-18 binding protein (IL- 18BP) compared to wild type IL-18 (WT IL-18) (SEQ ID NO: 701). In some embodiments,, wherein the IL- 18 polypeptide contains one or more amino acid substitutions that are located at residue positions selected from Y01, F02, E06, VI 1, C38, K53, D54, S55, T63, E69, K70, E85, C76, M86, T95, D98, and C127, wherein residue position numbering of the IL-18 polypeptides is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the one or more amino acid substitutions in the IL-18 polypeptide are selected from Y01G, F02A, E06K, VI II, C38S, C38A, K53A, D54A, S55A, T63A, C68S, C68A, E69C, K70C, C76S, C76A, E85C, T95C, D98C, C127S, and C127A. In some embodiments, the IL-18 polypeptide contains an E06K amino acid substitutions. In some embodiments, the IL-18 polypeptide contains a K53A amino acid substitution. In some embodiments, the IL-18 polypeptides contains a T63A amino acid substitution. In some embodiments, the IL-18 polypeptide comprises a VI II amino acid substitution. In some embodiments, the IL- 18 polypeptide comprises a substitution at one or more of residues C38, C68, C76, and C127. In some embodiments, the IL-18 polypeptide comprises substitutions at residues C38, C76, and C127 or residues C38, C68, C76, and C127. In some embodiments, each substitution at residues C38, C68, C76, and C127 is independently selected from a serine or alanine substitution. In some embodiments, the IL- 18 polypeptide comprisesWSGR Docket No. 56146-747.601C38A, C76A, and C127A substitutions, or wherein the IL-18 polypeptide comprises C38A, C68A, C76A, and C127A substitutions. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence described in Table 12, or wherein the IL-18 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 701-711.

[0010] In some embodiments, the IL-18 polypeptide is an activatable IL-18 polypeptide which comprises an artificial polypeptide comprising a protease cleavage site attached to the N-terminus of the IL- 18 polypeptide. In some embodiments, cleavage at the protease cleavage site enhances an IL- 18 related activity of the IL- 18 polypeptide. In some embodiments, the artificial polypeptide is of the formula: BM-CS, wherein BM is a blocking moiety and CS is a peptide comprising the protease cleavage site. In some embodiments, the blocking moiety is an IL- 18 propeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 731. In some embodiments, the IL-18 propeptide comprises the sequence set forth in SEQ ID NO: 731, 733, 735, or 736. In some embodiments, the protease cleavage site is cleaved by a protease which is found at higher concentrations and / or demonstrates higher proteolytic activity within the tumor microenvironment relative to non-tumor tissue. In some embodiments, the protease is selected from: kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), granzyme M, urokinase plasminogen activator (uPA), a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a matriptase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease. In some embodiments, the protease cleavage site is comprised in a protease recognition sequence having at least 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a peptide sequence set forth in Table 4. In some embodiments, the protease recognition sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 744, 745, 746, 747, 748, 749, 750, 752, 753, or 754. In some embodiments, the cleavage of the artificial polypeptide at the protease cleavage site leaves no amino acid residues of the artificial polypeptide attached to the IL- 18 polypeptide, or wherein cleavage of the artificial polypeptide at the protease cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the artificial polypeptide attached to the IL-18 polypeptide. In some embodiments, the artificial polypeptide comprises at least 2, 3, or 4 protease cleavage sites. In some embodiments, the IL-18 polypeptide comprises substitutions of one or more amino acids at or near the N- or C-terminus of the IL- 18 polypeptide which form part of a recognition sequence for the protease cleavage site. In some embodiments, the substitutions of the one or more amino acids at or near the N- or C-terminus of the IL- 18 polypeptide comprises amino acid substitutions of residues 1, 2, and / or 3 of the IL- 18 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence.WSGR Docket No. 56146-747.601

[0011] In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine exhibits an EC50 value for IFNv release in parental NK92 cells of at most about 10 nM, at most about 5 nM, at most about 2 nM, at most about 1 nM, at most about 0.5 nM, at most about 0.25 nM, or at most about 0.1 nM.

[0012] In some embodiments, the IL-18 polypeptide is in covalent association via a linker attached to a side chain of an amino acid residue of the IL- 18 polypeptide. In some embodiments, the linker is attached to residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, or 144 of the IL- 18 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the linker is attached to residue 68 of the IL- 18 polypeptide. In some embodiments, the linker is attached to an Fc region of the composition, preferably wherein the linker is attached to the Fc region at a position of a K246 amino acid residue, a K248 amino acid residue, a K288 amino acid residue, a K290 amino acid residue, or a K317 amino acid residue of the Fc region (EU numbering) (e.g., conjugation using AJICAP™ technology). In some embodiments, the linker is attached to the K248 residue.

[0013] In some embodiments, the IL-18 polypeptide is in covalent association via a C-terminal fusion of the IL-18 polypeptide to the portion of the composition to which it is attached, or the IL- 18 polypeptide is in covalent association via an N-terminal fusion of the IL- 18 polypeptide to the portion of the composition to which it is attached.

[0014] In some embodiments, the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1). In some embodiments, the first binding domain is comprised in an antigen binding fragment derived from an antibody. In some embodiments, the first binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3). In some embodiments, the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 1A, IB, or 1C. In some embodiments, the VH comprise an amino acid sequence of a VH set forth in Table 1 A, IB, or 1C. In some embodiments, the first binding domain is a VHH. In some embodiments, the first binding domain comprises: a) a VH CDR1 sequence of SEQ ID NO: 2; a VH CDR2 sequence of SEQ ID NO: 3; and a VH CDR3 sequence of SEQ ID NO: 4; b) a VH CDR1 sequence of SEQ ID NO: 6; a VH CDR2 sequence of SEQ ID NO: 7; and a VH CDR3 sequence of SEQ ID NO: 8; c) a VH CDR1 sequenceWSGR Docket No. 56146-747.601 of SEQ ID NO : 14; a VH CDR2 sequence of SEQ ID NO : 15 ; and a VH CDR3 sequence of CDR3 SEQ ID NO: 16; d) a VH CDR1 sequence of SEQ ID NO: 18; a VH CDR2 sequence of SEQ ID NO: 19; and a VH CDR3 sequence of CDR 3 of SEQ ID NO: 20; e) a VH CDR1 sequence of SEQ ID NO: 288, a VH CDR2 sequence of SEQ ID NO: 289, and a VH CDR3 sequence of SEQ ID NO: 290, or f) a VH CDR1, VH CDR2, and VHCDR3 of a VHH provided in Table 1C. In some embodiments, the VHH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1, 5, 13, 17, 287, or that of a VHH provided in Table 1C. In some embodiments, the VH comprises a) a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFD Y (SEQ ID NO: 82); b) a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88); or c) a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115). In some embodiments, the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 48, 50, or 76. In some embodiments, the first binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3). In some embodiments, the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the first binding domain is comprised. In some embodiments, the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 1A. In some embodiments, the VL comprises: a) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); b) a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); or c) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LAS YLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118). In some embodiments, the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 49, 51, or 77. In some embodiments, the first binding domain comprises: a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKNWSGR Docket No. 56146-747.601(SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); or b) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91; or c) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFD Y (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118) In some embodiments, the first binding domain comprises: a) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 49; b) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 50 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 51; c) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 76 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 77; d) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10; or e) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12. In some embodiments, the first binding domain is an scFv. In some embodiments, the first binding domain is a Fab.

[0015] In some embodiments, the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors. In some embodiments, the second binding domain is comprised in an antigen binding fragment derived from an antibody. In some embodiments, the second binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chainWSGR Docket No. 56146-747.601 second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3). In some embodiments, the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 2A, 2B, or 2C. In some embodiments, the VH comprise an amino acid sequence of a VH set forth in Table 2A, 2B, or 2C. In some embodiments, the VH comprises a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125). In some embodiments, the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 122. In some embodiments, the second binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3). In some embodiments, the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the second binding domain is comprised. In some embodiments, the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2A. In some embodiments, the VL comprises a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130). In some embodiments, the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 127. In some embodiments, the second binding domain comprises a VH having a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125), and a VL having a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130). In some embodiments, the second binding domain comprises a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 127. In some embodiments, theWSGR Docket No. 56146-747.601 second binding domain is an scFv. In some embodiments, the second binding domain is a Fab. In some embodiments, the second binding domain is a single domain antibody. In some embodiments, the second binding domain is a single domain heavy chain antibody (VHH). In some embodiments, the second binding domain comprises: a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204); b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208); c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFD Y (SEQ ID NO: 212); d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); e) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220); or f) a VH CDR1, VH CDR2, and VH CDR3 of a VHH provided in Table 2C. In some embodiments, the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, or 280-284, or the a VH sequence provided in Table 2C. In some embodiments, the second binding domain comprises a light chain single domain antibody. In some embodiments, the second binding domain comprises a VL CDR1 having the sequence RASQWIGPELS (SEQ ID NO: 223), a VL CDR2 having the sequence HTSILQS (SEQ ID NO: 224), and a VL CDR3 having the sequence QQYMFQPRT (SEQ ID NO: 225). In some embodiments, the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 222. In some embodiments, the second binding domain is an anti-VEGFA anticalin. In some embodiments, the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 226.

[0016] In some embodiments, the composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain. In some embodiments, the Fc domain is derived from an IgG. In some embodiments, the Fc domain is derived from an IgGl or IgG4.WSGR Docket No. 56146-747.601 cX-Y-Z X'-Y'-Z’ i

[0017] In some embodiments, the composition comprises a structure of the formula:c' wherein: Y is the first CH2 and CH3 domains; Y’ is the second CH2 and CH3 domains; X and X’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, the cytokine, a copy of the cytokine, or absent; Z and Z’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, the cytokine, a copy of the cytokine, a third binding domain targeting PD-1, or a fourth binding domain targeting VEGFA, or absent. C and C’ are each independently the cytokine, a copy of the cytokine, or absent, wherein C and C’, if present, are attached to a side chain of a residue of the Fc domain via a linker; wherein X, Y, and Z and X’, Y’ and Z’ are depicted in an N-terminal to C-terminal direction; and wherein each of X, X’, Z, and Z’ are independently and optionally connected to Y or Y’ via a peptide linker. In some embodiments, X is the first binding domain; X’ is a copy of the first binding domain, the cytokine, or absent; one of Z or Z’ is the second binding domain and the other is absent or a copy of the second binding domain; and one of C or C’ is the cytokine and the other is absent, or both C and C’ are absent. In some embodiments, X’ is the cytokine and C and C’ are both absent. In some embodiments, X’ is the copy of the first binding domain and one of C or C’ is the cytokine. In some embodiments,: Z is the second binding domain and Z’ is a copy of the second binding domain; Z is the second binding domain and Z’ is absent; or Z is absent and Z’ is the second binding domain. In some embodiments, X is the second binding domain, X’ is a copy of the second binding domain, the cytokine, or absent; one of Z or Z’ is the first binding domain and the other is absent or a copy of the first binding domain; and one of C or C’ is the cytokine and the other is absent, or both C and C’ are absent. In some embodiments, X’ is the cytokine and C and C’ are both absent. In some embodiments, X’ is a copy of the second binding domain one of C or C’ is the cytokine. In some embodiments,: Z is the first binding domain and Z’ is a copy of the first binding domain; Z is the first binding domain and Z’ is absent; or Z is absent and Z’ is the first binding domain. In some embodiments, X is the first binding domain, X’ is the second binding domain, one of C or C’ is the cytokine and the other is absent, Z is absent or the third binding domain, and Z’ is absent or the fourth binding domain. In some embodiments,: Z is the third binding domain and Z’ is absent; Z is absent and Z’ is the fourth binding domain; or both Z and Z’ are absent. In some embodiments, if the third binding domain is present, the first binding domain comprises a Fab and the third binding domain comprises an scFv, and wherein the Fab and the scFv comprise the same VH and VL. In some embodiments, if the fourth binding domain is present, the second binding domain comprises a Fab and the fourth binding domain comprisesWSGR Docket No. 56146-747.601 an scFv, and wherein the Fab and the scFv comprise the same VH and VL. In some embodiments, X is one of the first or second binding domains and is a Fab, VHH, or scFv. In some embodiments, X is one of the first or second binding domains and is a Fab. In some embodiments, X’ is a copy of X. In some embodiments, one Z or Z’ is one of the first or second binding domains and is an scFv or a VHH, wherein if X is the first binding domain then Z or Z’ is the second binding domain and if X is the second binding domain then Z or Z’ is the first binding domain. In some embodiments,: X is a Fab and the first binding domain, X’ is a copy of the first binding domain, Z is an scFv or VHH and is the second binding domain, Z’ is a copy of the second binding domain, C is the cytokine, and C’ is absent; or X is a Fab and the second binding domain, X’ is a copy of the second binding domain, Z is an scFv or VHH and is the first binding domain, Z’ is a copy of the first binding domain, C is the cytokine, and C’ is absent; or X is a Fab and is the first binding domain, X’ is a Fab and is the second binding domain, Z and Z’ are absent, and C or C’ is the cytokine and the other is absent; X is a Fab and is the first binding domain, X’ is a Fab and is the second binding domain; Z is an scFv or VHH and is the third binding domain, Z’ is absent, and C or C’ is the cytokine and the other is absent; or X is a Fab and is the second binding domain, X’ is a Fab and is the first binding domain, Z is an scFv or VHH and is the fourth binding domain, Z’ is absent, and C or C’ is the cytokine and the other is absent; or X is a Fab and is the first binding domain, X’ is the cytokine, Z is an scFv or VHH and is the second binding domain, Z’ is a copy of the second binding domain, and C and C’ are both absent; or X is a Fab and is the second binding domain, X’ is the cytokine, Z is an scFv or VHH and is the first binding domain, Z’ is a copy of the first binding domain, and C and C’ are both absent; or X is a Fab and is the first binding domain, X’ is an scFv and is the second binding domain; Z and Z’ are both absent, and C or C’ is the cytokine and the other is absent; or X is a Fab and is the second binding domain, X’ is an scFv and is the first binding domain, Z and Z’ are both absent, and C or C’ is the cytokine and the other is absent. In some embodiments, the composition comprises only one cytokine. In some embodiments, the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 229-234 or 236-241.

[0018] In some embodiments, the first binding domain comprises a Fab, and wherein the composition comprises: a) a first polypeptide chain comprising the VL of the first binding domain; b) a second polypeptide chain comprising the VH of the first binding domain; and c) a third polypeptide chain comprising the IL-2 polypeptide.

[0019] In some embodiments, the first binding domain comprises: a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VLWSGR Docket No. 56146-747.601 having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); or b) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); or c) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFD Y (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118). In some embodiments, the first binding domain comprises: a) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 49; b) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 50 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 51; or c) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 76 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 77. In some embodiments, the first polypeptide chain comprises, in N- to C-terminal direction, the VL and light chain constant region. In some embodiments, the light chain constant region comprises an amino acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 276. In some embodiments, the first polypeptide chain comprises the sequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL S STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC . I

[0020] n some embodiments, the second polypeptide chain comprises, in an N-terminal to C- terminal direction, the VH of the Fab of the first binding domain and an antibody constant region. In some embodiments, the antibody constant region is an IgGl or IgG4 constant region. In some embodiments, the antibody constant region comprises, in an N-terminal to C-terminal direction, a CHI domain, a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, a) theWSGR Docket No. 56146-747.601CHI domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 272-275; b) the hinge region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 265-271; and / or c) the CH2 and CH3 domains together comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 229-234. In some embodiments, the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab, the antibody constant region, an optional peptide linker, and the second binding domain. In some embodiments, the optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n (SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, the second binding domain is a VHH. In some embodiments, the VHH comprises a two proline peptide on its C-terminus. In some embodiments, the VHH comprises a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204); b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208); c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFDY (SEQ ID NO: 212); d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); or e) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRL AD TYEY (SEQ ID NO: 220). In some embodiments, the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, or 280-284. In some embodiments, the VHH comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO. 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220). In some embodiments, the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:200, 217, or 221. In some embodiments, the second polypeptide chain further comprises an additional binding domain targeting VEGFA. In some embodiments, the additional bindingWSGR Docket No. 56146-747.601 domain targeting VEGFA is a VHH. In some embodiments, both the second binding domain and the additional binding domain targeting VEGFA are both VHHs comprising an identical amino acid sequence, or wherein the VHH positioned C-terminal to the other VHH comprises an additional two proline peptide on its C-terminus as compared to the other VHH, optionally wherein the VHH comprises a two proline peptide on its C-terminus. In some embodiments, the additional binding domain targeting VEGFA comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG ( SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220). In some embodiments, the additional binding domain targeting VEGFA an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200, 217, or 221. In some embodiments, the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab, an antibody constant region, an optional peptide linker, the second binding domain, a second optional peptide linker, and the additional binding domain targeting VEGFA. In some embodiments, the second optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n (SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, the second polypeptide chain comprises the sequence set forth in any one of SEQ ID NOs: 163, 164, 169, 171, or 172.

[0021] In some embodiments, the third polypeptide chain comprises, in an N-terminal to C- terminal direction, the IL-18 polypeptide, an optional peptide linker, and an antibody constant region. In some embodiments, the optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, the third polypeptide chain comprises, in an N-terminal to C-terminal direction, the IL-2 polypeptide, the optional peptide linker, an antibody constant region, a second optional peptide linker, and the second binding domain or an additional binding domain targeting VEGFA, wherein the additional binding domain targeting VEGFA is present if the second binding is present on the second polypeptide chain or if the second binding domain is also present on the third polypeptide chain. In some embodiments, the second optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n (SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9,WSGR Docket No. 56146-747.601 or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, the second binding domain or the additional binding domain targeting VEGFA is a VHH. In some embodiments, the VHH comprises a two proline peptide on its C-terminus. In some embodiments, the second binding domain or the additional binding domain targeting VEGFA comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220). In some embodiments, the second binding domain or the additional binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200, 217 or 221. In some embodiments, the third polypeptide comprises the second binding domain and the additional binding domain targeting VEGFA separated by an optional peptide linker. In some embodiments, optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4 (SEQ ID NO: 30). In some embodiments, both the second binding domain and the additional binding domain targeting VEGFA are both VHHs comprising an identical amino acid sequence, or wherein the VHH positioned C-terminal to the other VHH comprises an additional two proline peptide on its C-terminus as compared to the other VHH. In some embodiments, the antibody constant region is an IgGl or IgG4 constant region, or a portion thereof. In some embodiments, the antibody constant region comprises, in an N-terminal to C- terminal direction, a hinge region, a CH2 domain, and a CH3 domain. In some embodiments, the antibody constant region comprises, in an N-terminal to C-terminal direction, a hinge region portion, a CH2 domain, and a CH3 domain. In some embodiments, a) the hinge region portion comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 265-271; and / or b) the CH2 and CH3 domains together comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 229-234. In some embodiments, the third polypeptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 722 or 723.

[0022] In some embodiments,: the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 47; the second polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 163, 164, 169, 171, or 172; and the third polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 722 or 723. In some embodiments, the firstWSGR Docket No. 56146-747.601 polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, the amino acid sequences set forth in: SEQ ID NOs: 47, 163, and 722;SEQ ID NOs: 47, 163, and 723; SEQ ID NOs: 47, 164, and 722;SEQ ID NOs: 47, 164, and 723; SEQ ID NOs: 47, 169, and 722;SEQ ID NOs: 47, 169, and 723; SEQ ID NOs: 47, 171, and 722;SEQ ID NOs: 47, 171, and 723; SEQ ID NOs: 47, 172, and 722; orSEQ ID NOs: 47, 172, and 723. In some embodiments, the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 47, 164, and 722.

[0023] In another aspect provided herein is an immunocytokine composition comprising: a binding domain targeting vascular endothelial growth factor A (VEGFA); and an IL-18 polypeptide. In some embodiments, the IL-18 polypeptide is any of the IL-18 polypeptides described herein. In some embodiments, the binding domain targeting VEGFA is any of those described herein. In some embodiments, the binding domain targeting VEGFA is an antibody or an antigen binding fragment thereof. In some embodiments, the binding domain targeting VEGFA is a monoclonal antibody. In some embodiments, the composition further comprises a binding domain targeting PD-1. In some embodiments, the binding domain targeting PD-1 is any one of the binding domains targeting PD-1 described herein.

[0024] In an aspect, the present disclosure provides one or more polynucleotides encoding the composition of any of the embodiments disclosed herein, or a portion thereof.

[0025] In an aspect, the present disclosure provides a host cell comprising a composition disclosed herein or a polynucleotide disclosed herein (e.g., one or more polynucleotides encoding the composition of any of the embodiments disclosed herein, or a portion thereof).

[0026] In an aspect, the present disclosure provides a pharmaceutical composition comprising the composition of any of the embodiments disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0027] In an aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.INCORPORATION BY REFERENCE

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict theWSGR Docket No. 56146-747.601 disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIGURE 1A shows an exemplary embodiment of a multifunctional immunocytokine as described herein, which comprises an IL-18 polypeptide conjugated to an Fc domain, said Fc domain linked to two Fabs targeting VEGF and two scFvs targeting PD-1.

[0030] FIGURE IB shows an analogous multifunctional immunocytokine to that of FIG. 1A, but with an activatable IL-18 polypeptide depicted with its mask intact. The mask is linked to the IL-18 polypeptide by a protease cleavable linker. Upon cleavage of the protease cleavable linker, the mask is able to dissociate, thus allowing the IL-18 polypeptide to bind with the IL-18 receptor and signal.

[0031] FIGURES 2A-2D depict formats of Fc domain containing constructs which contain anti- VEGFA and anti -PD-1 binding domains which can be conjugated with a cytokine such as an IL- 18 polypeptide to provide an immunocytokine composition according to the instant disclosure. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or derivatives thereof. In some embodiments, the anti -PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab or Nivolumab, or derivatives thereof. In some embodiments, such as those depicted in FIG. 1C and FIG. ID, an Fc domain containing construct comprises a single domain antibody (e.g., a VHH or light chain single domain antibody) as one of the binding domains (e.g., the anti -PD-1 or anti-VEGFA binding domain).

[0032] FIGURE 3A-3D depict formats of asymmetric Fc domain containing constructs contain anti-VEGFA and anti-PD-1 binding domains which can be conjugated with a cytokine such as an IL-18 polypeptide to provide an immunocytokine composition according to the instant disclosure. These constructs contain a K248A substitution which facilitate the conjugation of a single cytokine to the construct (e.g., by AJICAP™ technology), thus readily providing an immunocytokine composition which contains only one cytokine attached. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab. In some embodiments, such as those depicted in FIG. 3C and FIG. 3D, the Fc domain containing construct comprises a single domain antibody (e.g., a VHH or light chain single domain antibody) as one of the binding domains (e.g., the anti-PD-1 or anti-VEGFA binding domain). In some embodiments, the anti-VEGFA single domain antibody binding domain is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs: 201, 205, 209, 213, 217, 221, 222, 280-284 or a variant single domain antibody which comprises the CDRs set forth in one of those sequences). In some embodiments, the anti-PD-1 single domainWSGR Docket No. 56146-747.601 antibody binding domain is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs: 1, 5, 13, 17, 287, or a VHH provided in Table 1C (e.g., VHH47) or a variant single domain antibody which comprises the CDRs set forth in one of those sequences).

[0033] FIGURES 4A-4C depict formats of asymmetric Fc domain containing constructs in bispecific antibody formats containing anti-VEGFA and anti-PD-1 binding domains which can be conjugated with a cytokine such as an IL- 18 polypeptide to provide an immunocytokine composition according to the instant disclosure. In the formats depicted, light chain pairing technology must be utilized during manufacture to ensure proper pairing of VH and VL domains in the construct. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or a derivative thereof. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab, or a derivative thereof. In some embodiments, such as that depicted in FIG. 4B, a second anti-VEGFA binding domain is incorporated via fusion to the C-terminus (optionally through a peptide linker) of the Fc domain. The second anti-VEGFA binding domain depicted in FIG. 4B is an scFv. In some embodiments, the second anti-VEGFA binding domain is derived from the same antibody as the first anti-VEGFA binding domain (e.g., the second anti-VEGFA binding domain contains the same VH and VL as the first anti-VEGFA binding domain). A similar embodiment is also depicted in FIG. 4C, which depicts a construct in which the second VEGFA binding domain is a single domain antibody (e.g., a VHH). In some embodiments, the single domain antibody is one of those described herein (e.g., a single domain antibody of any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, 222, 280-284, or a variant single domain antibody which comprises the CDRs set forth in one of those sequences).

[0034] FIGURES 5A and 5B depict formats of asymmetric Fc domain containing constructs in bispecific antibody formats containing anti-VEGFA and anti-PD-1 binding domains which can be conjugated with a cytokine such as an IL- 18 polypeptide to provide an immunocytokine composition according to the instant disclosure. This bispecific antibody format utilizes an scFv as one of the binding domains and eliminates the need for light chain pairing technology to be used in their manufacture. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab, or derivatives thereof.

[0035] FIGURES 6A and 6B depict formats of immunocytokine compositions according to the instant disclosure in which an IL-18 polypeptide is fused to the Fc domain. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or derivatives thereof. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM- 009, Pembrolizumab, or Nivolumab, or derivatives thereof.WSGR Docket No. 56146-747.601

[0036] FIGURES 7A, 7B, and 7C depict formats of immunocytokine compositions according to the instant disclosure in which an IL-18 polypeptide is fused to the Fc domain. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, or derivatives thereof. In some embodiments, the anti-PD-1 binding domains comprise the VH and VL of LZM-009, Pembrolizumab, or Nivolumab, or derivatives thereof.

[0037] FIGURES 8A-8E depict formats of immunocytokine compositions according to the instant disclosure in which an IL-18 polypeptide is fused to an Fc domain and in which the constructs comprise VHH domains as at least one of the binding domains.

[0038] FIGURES 9A-9C depict traces from heparin chromatography performed on immunocytokines according to the instant disclosure. The tested immunocytokines are Compositions 1, 3, and 8, respectively.

[0039] FIGURES 10A-10D depicted results from experiments testing the ability of immunocytokines described herein to stimulate IFNg secretion in parental and PD1+NK92 cells.

[0040] FIGURE 11 shows results from an AlphaLISA experiment measuring the ability of immunocytokines described herein to bind to IL-18 binding protein (IL-18BP).

[0041] FIGURES 12A-12C show results from experiments testing the ability of immunocytokines described herein to inhibit the interaction of PD-1 with PD-L1 in the presence or absence of VEGF.

[0042] FIGURE 13 shows average tumor volumes in C57BL / 6 mice transgenic for human PD-1 bearing MC38 tumors after administration of the indicated Compositions..

[0043] FIGURE 14 average tumor volumes in individual C57BL / 6 mice transgenic for human PD-1 bearing MC38 tumors after administration of the indicated Compositions

[0044] FIGURE 15 depicts survival curves for C57BL / 6 mice transgenic for human PD-1 bearing MC38 tumors after treatment with the indicated Compositions.DETAILED DESCRIPTION

[0045] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this present disclosure, which are encompassed within its scope.

[0046] Although various features of the present disclosure may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.WSGR Docket No. 56146-747.601

[0047] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Multifunctional Immunocytokines

[0048] Provided herein in an aspect are multifunctional immunocytokines. In some embodiments, the multifunctional immunocytokines comprise a PD-1 binding domain, a VEGFA binding domain, and a cytokine, such as IL- 18.

[0049] In some instances, it is desirable that the IL- 18 polypeptide is one which is either activatable (e.g., has a null or low IL- 18 related activity until activation, such as cleavage of a masking group by a tumor microenvironment protease, such as those described in PCT Pub. No. WO2023161853A1 (corresponding US Pat. Pub. No. US20240116997A1)) or one which is detuned (i.e., less potent / active) as compared to wild type IL-18 in order to provide a high degree of safety and low toxicity / side effects. In some embodiments, the IL-18 polypeptide is only modestly detuned compared to wild type IL-18. In some instances, a fully potent IL-18 polypeptide (e.g., an IL- 18 polypeptide having IL-18 receptor related activity greater than or equal to that of wild type IL- 18) can be used.Binding Domains

[0050] An immunocytokine composition described herein comprises one or more binding domains which specifically target one of PD-1 or VEGFA. In some preferred embodiments, an immunocytokine composition comprises both PD-1 and VEGFA binding domains.

[0051] A binding domain selectively binds or preferentially binds to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. As such, “specific binding” or “preferential binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to specific binding means preferential binding where the affinity of the binding domain is at least at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7- fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the affinity of the binding domain for unrelated amino acid sequences.

[0052] In some embodiments, a binding domain of the disclosure can inhibit the action / activity of the substance to which it binds (e.g., by disruption an interaction of PD-1 with its receptor (e.g., PD-L1) and / or by disruption of VEGFA with one or more of its receptors (e.g., VEGFR1, VEGFR2, VEGFR3).WSGR Docket No. 56146-747.601

[0053] The binding domains of the instant disclosure can be of any desired format which binds specifically to the intended target. As such, the nature of the binding domains is not necessarily limited and can encompass, for example, polypeptides (e.g., antigen binding fragments derived from antibodies or other peptides which bind specifically for the target, such as variants of receptors of the targets or other polypeptides), aptamers (e.g., nucleic acid aptamers), small molecules, and the like. In some embodiments, a binding domain of the instant disclosure is a polypeptide.

[0054] In some embodiments, a binding domain of the instant disclosure comprises an antigen binding fragment derived from an antibody, or a variant thereof. Antigen binding fragments of antibodies, including any of the antibodies herein (e.g., the anti-PD-1 antibodies or anti-VEGFA antibodies described below), are contemplated as being used as binding domains in immunocytokine compositions described herein. The terms “antigen binding portion of an antibody,” “antigen binding domain,” “antibody fragment,” or a “functional fragment of an antibody” are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. In some embodiments, such regions refer to the VH and / or the VL of the antibody, or a derivative or portion thereof. Representative antigen binding fragments include, but are not limited to, a Fab, a Fab', a F(ab')2, a bispecific F(ab')2, a trispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a dsFv, a bispecific scFv, a variable heavy domain, a variable light domain, a variable NAR domain, bispecific scFv, an AVIMER®, a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a minibody, a maxibody, a camelid, a VHH, a minibody, an intrabody, fusion proteins comprising an antibody portion (e.g., a domain antibody), a single chain binding polypeptide, a scFv-Fc, a Fab-Fc, a bispecific T cell engager (BiTE; two scFvs produced as a single polypeptide chain, where each scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a tetravalent tandem diabody (TandAb; an antibody fragment that is produced as a non-covalent homodimer folder in a head-to-tail arrangement, e.g., a TandAb comprising an scFv, where the scFv comprises an amino acid sequences a combination of CDRs or a combination of VL / VL described herein), a Dual-Affinity Re-targeting Antibody (DART; different scFvs joined by a stabilizing interchain disulphide bond), a bispecific antibody (bscAb; two single-chain Fv fragments joined via a glycine-serine linker), a single domain antibody (sdAb), a fusion protein, a bispecific disulfide-stabilized Fv antibody fragment (dsFv-dsFv'; two different disulfide-stabilized Fv antibody fragments connected by flexible linker peptides).

[0055] In some embodiments, a binding domain of the instant disclosure is a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, aWSGR Docket No. 56146-747.601 tetrabody, a maxibody, a cam elid, a single domain antibody (e.g., a VHH), a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a cam elid, a single domain antibody (e.g., a VHH), a Fab-Fc, or a scFv-Fc. In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, F(ab')2, abispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), a camelid, or a single domain antibody (e.g., a VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), a camelid, or a single domain antibody (e.g., a VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a Fab’, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), or a single domain antibody (e.g., a VHH). In some embodiments, a binding domain of the instant disclosure is Fab, a single chain variable fragment (scFv), or a single domain antibody (e.g., a VHH). In some embodiments, each binding domain of an immunocytokine composition is independently one of those described above. In some embodiments, each binding domain of an immunocytokine composition described herein is independently a Fab, an scFv, or a single domain antibody (e.g., a VHH).

[0056] In some embodiments, a binding domain of the instant disclosure comprises a VH derived from an antibody, or a derivative thereof. In some embodiments, the VH retains the CDRs of the VH of the antibody from which the VH of the immunocytokine composition is derived (e.g., a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3)). In some embodiments, the VH retains the CDRS of the VH of the antibody from which it is derived and comprises one or more mutations in the framework region. In some embodiments, the VH retains the CDRs of the VH of the antibody from which it is derived and comprises up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in the framework region. In some embodiments, each mutation is a conservative mutation. In some embodiments, the binding domain comprises a VH of a corresponding antibody (e.g., with no mutations to the framework region). In some embodiments, the VH is comprises in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or abispecific antibody. In some embodiments, the VH is comprised in a Fab, a Fab’, an scFv, or a VHH. In some embodiments, the VH isWSGR Docket No. 56146-747.601 comprised in a Fab, an scFv, or a VHH. In some embodiments, the VH is comprised in a Fab or an scFv. In some embodiments, the VH is a VHH.

[0057] In some embodiments, a VHH of the instant disclosure can comprise one or more modifications which improve immunogenicity or reduce binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acids Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP, KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., LI IK or LI IV) and / or Leu 89 (e.g., L89T), and Lin et al., “A structurebased engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944. doi.org / 10.1371 / joumal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C-terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, a VHH described herein comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, a VHH described herein comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two VHHs are linked in series (e.g., by a flexible peptide linker), in some instance only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C-terminal “SS” of each VHH).

[0058] In some embodiments, a binding domain of the instant disclosure comprises a VL derived from an antibody, or a derivative thereof. In some embodiments, the VL retains the CDRs of the VL of the antibody from which the VL of the immunocytokine composition is derived (e.g., a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3)). In some embodiments, the VL retains the CDRS of the VL of the antibody from which it is derived and comprises one or more mutations in the framework region. In some embodiments, the VL retains the CDRs of the VL of the antibody from which it is derived and comprises up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutations in the framework region. In some embodiments, each mutation is a conservative mutation. In some embodiments, the binding domain comprises a VL of a corresponding antibody (e.g., with no mutations to the framework region). In some embodiments, the VL is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2,WSGR Docket No. 56146-747.601 a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VL is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VL is comprised in a Fab or an scFv. In some embodiments, the VL is comprised in the same Fab, Fab’, F(ab’)2, bispecific F(ab’)2, scFv, bispecific scFv, dsFv, minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody as the corresponding VH. In some embodiments, the VL is comprised in the same Fab, Fab’, or scFv as the corresponding VH. In some embodiments, the VL is comprised in the same Fab or scFv as the corresponding VH.

[0059] In some embodiments, a binding domain of the instant disclosure is a light chain single domain antibody.

[0060] In some embodiments, a binding domain specifically binds to one or more epitopes on one or more target antigens. In some embodiments, a binding domain selectively binds to an epitope on a single antigen.PD-1 Targeting Binding Domains

[0061] In some embodiments, an immunocytokine composition of the instant disclosure comprise one or more binding domains which target programmed cell death protein 1 (PD-1). In some embodiments, a binding domain incorporated into an immunocytokine composition of the disclosure specifically binds to PD-1. In some embodiments, the anti-PD-1 binding domain is capable of disrupting and / or preventing the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

[0062] Programmed cell death protein 1 (also known as PD-1 and CD279), is a cell surface receptor that plays a role in down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune cell inhibitory molecule that is expressed on activated B cells, T cells, and myeloid cells. PD-1 represents an immune checkpoint and guards against autoimmunity via a dual mechanism of promoting apoptosis (programmed cell death) in antigen-specific T-cells in lymph nodes while reducing apoptosis in regulatory T cells. PD-1 is a member of the CD28 / CTLA-4 / ICOS costimulatory receptor family that delivers negative signals that affect T and B cell immunity. PD-1 is monomeric both in solution as well as on cell surface, in contrast to CTLA-4 and other family members that are all disulfide-linked homodimers. Signaling through the PD-1 inhibitory receptor upon binding its ligand, PD-L1, suppresses immune responses against autoantigens and tumors and plays a role in the maintenance of peripheral immune tolerance. The interaction between PD-1 and PD-L1 results in a decrease in tumor infiltrating lymphocytes, a decrease in T cell receptor mediated proliferation, and immune evasion by the cancerous cells. A non-limiting, exemplary, human PD-1 amino acid sequence isWSGR Docket No. 56146-747.601MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL (SEQ ID NO: 31).

[0063] In some embodiments, the anti-PD-1 binding domain is comprised in an antigen binding fragment derived from an antibody. For example, the anti-PD-1 binding domain can be derived from any anti-PD-1 antibody known in the art or which can be made according to well understood methods. In some embodiments, the antibody or antigen binding fragment thereof of an immunocytokine composition described herein is derived from an anti-PD-1 antibody or antigen binding fragment.

[0064] In one embodiment, an anti-PD-1 binding domain of an immunocytokine composition comprises and antigen binding fragment. In some embodiments, the anti-PD-1 binding domain of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein, or of other anti-PD-1 antibodies or antigen binding fragments known in the art. In another embodiment, an anti-PD-1 antibody or an anti-PD-1 antigen binding fragment of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein or of other anti-PD-1 antibodies or antigen binding fragments known in the art.

[0065] In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the CDRs of an antibody selected from Tislelizumab, Baizean, 0KVO411B3N, BGB-A317, hu317- l / IgG4mt2, Sintilimab, Tyvyt, IBI-308, Toripalimab, TeRuiPuLi, Terepril, Tuoyi, JS-001, TAB- 001, Camrelizumab, HR-301210, INCSHR-01210, SHR-1210, Cemiplimab, Cemiplimab-rwlc, LIBTAYO®, 6QVL057INT, H4H7798N, REGN-2810, SAR-439684, Avelumab, BAVENCIO®, 451238, KXG2PJ551I, MSB-0010682, MSB-0010718C, PF-06834635, Durvalumab, IMFINZI®, 28X28X9OKV, MEDI-4736, Lambrolizumab, Pembrolizumab, KEYTRUDA®, MK-3475, SCH-900475, h409Al l, Nivolumab, Nivolumab BMS, OPDIVO®, BMS-936558, MDX-1106, ONO-4538, Prolgolimab, Forteca, BCD-100, Penpulimab, AK-105, Zimberelimab, AB-122, GLS-010, WBP-3055, Balstilimab, 1Q2QT5M7EO, AGEN-2034, AGEN-2034w, Genolimzumab, Geptanolimab, APL-501, CBT-501, GB-226, Dostarlimab, ANB-011, GSK- 4057190A, P0GVQ9A4S5, TSR-042, WBP-285, Serplulimab, HLX-10, CS-1003, Retifanlimab, 2Y3T5IF01Z, INCMGA-00012, INCMGA-0012, MGA-012, Sasanlimab, LZZ0IC2EWP, PF- 06801591, RN-888, Spartalizumab, NVP-LZV-184, PDR-001, QOG25L6Z8Z,Relatlimab / nivolumab, BMS-986213, Cetrelimab, JNJ-3283, JNJ-63723283, LYK98WP91F,WSGR Docket No. 56146-747.601Tebotelimab, MGD-013, BCD-217, BAT-1306, HX-008, MEDI-5752, JTX-4014, Cadonilimab, AK-104, BI-754091, Pidilizumab, CT-011, MDV-9300, YBL-006, AMG-256, RG-6279, RO- 7284755, BH-2950, IBI-315, RG-6139, RO-7247669, ONO-4685, AK-112, 609-A, LY-3434172, T-3011, MAX-10181, AMG-404, IBI-318, MGD-019, INCB-086550, ONCR-177, LY-3462817, RG-7769, RO-7121661, F-520, XmAb-23104, Pd-l-pik, SG-001, S-95016, Sym-021, LZM-009 (a.k.a., Lipustobart), Budigalimab, 6VDO4TY3OO, ABBV-181, PR-1648817, CC-90006, XmAb-20717, 2661380, AMP-224, B7-DCIg, EMB-02, ANB-030, PRS-332,[89Zr]Deferoxamide-pembrolizumab, 89Zr-Df-Pembrolizumab, [89Zr]Df-Pembrolizumab, STI- 1110, STI-Al l 10, CX-188, mPD-1 Pb-Tx, MCLA-134, 244C8, ENUM 224C8, ENUM C8, 388D4, ENUM 388D4, ENUM D4, MEDI0680, or AMP-514 incorporated into a VH and VL. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of any one of these antibodies. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv- Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0066] An anti- PD-1 binding domain can comprise a heavy chain, a VH, or a VH-CH1 domain (e.g., as in a Fab) having an amino acid sequence of any one of those set forth in Table 1 A, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-PD-1 binding domain can comprise (or further comprise) a light chain (e.g., as in a Fab) or VL having an amino acid sequence of any one of those described in Table 1A, or a portion corresponding to a VL thereof. In some embodiments, the heavy chain, VH, or VH-CH1 domain and VL or light chain are from the same antibody or antigen binding fragment described in Table 1 A.

[0067] In some embodiments, an anti-PD-1 binding domain of the disclosure comprises the CDRs (e.g., VH and VL CDRs) of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab,WSGR Docket No. 56146-747.601Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab incorporated into a VH and VL. In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the VH and VL of Tislelizumab, Sintilimab, Toripalimab, Terepril, Camrelizumab, Cemiplimab, Pembrolizumab Nivolumab, Prolgolimab, Penpulimab, Zimberelimab, Balstilimab, Genolimzumab, Geptanolimab, Dostarlimab, Serplulimab, Retifanlimab, Sasanlimab, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab, Pidilizumab, LZM-009 (a.k.a. Lipustobart), or Budigalimab. In some embodiments, the anti-PD- 1 binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0068] In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab, Pembrolizumab, LZM-009, Dostarlimab, Sintilimab, Spartalizumab, Tislelizumab, or Cemiplimab. In some embodiment, the anti-PD-1 binding domain comprises the VH and VL of Dostarlimab, Sintilimab, Spartalizumab, or Tislelizumab. In some embodiments, the anti-PD- 1 binding domain comprises the VH and VL of Nivolumab, Pembrolizumab, LZM-009 (a.k.a. Lipustobart), or Cemiplimab. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of Nivolumab in a Fab or scFv format. In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of LZM-009 (a.k.a. Lipustobart). In some embodiments, the anti-PD-1 binding domain comprises the VH and VL of LZM-009 (a.k.a. Lipustobart) in a Fab or scFv format.

[0069] TABLES 1A, IB, and 1C provide the sequences of exemplary anti-PD-1 antibodies and anti-PD-1 antigen binding fragments which contain sets of CDRs which can be incorporated into VHs and / or VLs and used as anti-PD-1 binding domains as described herein. In some embodiments, the VHs and VLs of the antibodies in Table 1A are incorporated into anti-PD-1 binding domains (e.g., in a Fab or scFv format). In some embodiments, CDRs of a VH describedWSGR Docket No. 56146-747.601 in Table 1 A, IB, or 1C are incorporated into a binding domain as a VHH. In some embodiments, a VH as described in Table 1 A, IB, or 1C is incorporated into a binding domain as a VHH.

[0070] In some instances, the SEQ ID NOs listed in Table 1 A contain full-length heavy or light chains of the indicated antibodies with the VH or VL respectively indicated in bold. Where there is a reference herein to a VH or VL of a SEQ ID NO in Table 1 A which contains a full-length heavy or light chain, it is intended to reference the bolded portion of the sequence. For example, reference to “a VH having an amino acid sequence shown in SEQ ID NO: 32” refers to the bolded portion of SEQ ID NO: 32 in Table 1 A.

[0071] An anti-PD-1 binding domain can comprise a VH having an amino acid sequence of any one of SEQ ID NOS: 32, 34, 36, 38, 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. An anti-PD-1 binding domain can comprise (or further comprise) a VL having an amino acid sequence of any one of SEQ ID NOS: 33, 35, 37, 39, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79.

[0072] An anti-PD-1 binding domain can comprise a heavy chain or VH having an amino acid sequence of any one of SEQ ID NOS: 32, 34, 36, 38, 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-PD-1 binding domain can comprise (or further comprise) a light chain or VL having an amino acid sequence of any one of SEQ ID NOS: 33, 35, 37, 39, 41, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 79, or a portion corresponding to a VL thereof.

[0073] In one instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 32, and a VL having an amino acid sequence shown in SEQ ID NO: 33. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 34, and a VL having an amino acid sequence shown in SEQ ID NO: 35. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 36, and a VL having an amino acid sequence shown in SEQ ID NO: 37. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 38, and a VL having an amino acid sequence shown in SEQ ID NO: 39. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 40, and a VL having an amino acid sequence shown in SEQ ID NO: 41. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 46, and a VL having an amino acid sequence shown in SEQ ID NO: 47. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 48, and a VL having an amino acid sequence shown in SEQ ID NO: 49. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acidWSGR Docket No. 56146-747.601 sequence shown in SEQ ID NO: 50, and a VL having an amino acid sequence shown in SEQ ID NO: 51. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 52, and a VL having an amino acid sequence shown in SEQ ID NO: 53. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 54, and a VL having an amino acid sequence shown in SEQ ID NO: 55. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 56, and a VL having an amino acid sequence shown in SEQ ID NO: 57. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 58, and a VL having an amino acid sequence shown in SEQ ID NO: 59. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 60, and a VL having an amino acid sequence shown in SEQ ID NO: 61. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 62, and a VL having an amino acid sequence shown in SEQ ID NO: 63. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 64, and a VL having an amino acid sequence shown in SEQ ID NO: 65. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 66, and a VL having an amino acid sequence shown in SEQ ID NO: 67. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 68, and a VL having an amino acid sequence shown in SEQ ID NO: 69. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 70, and a VL having an amino acid sequence shown in SEQ ID NO: 71. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 72, and a VL having an amino acid sequence shown in SEQ ID NO: 73. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 74, and a VL having an amino acid sequence shown in SEQ ID NO: 75. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 76, and a VL having an amino acid sequence shown in SEQ ID NO: 77. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 78, and a VL having an amino acid sequence shown in SEQ ID NO: 79. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 9, and a VL having an amino acid sequence shown in SEQ ID NO: 10. In another instance, an anti-PD-1 binding domain comprises a VH having an amino acid sequence shown in SEQ ID NO: 11, and a VL having an amino acid sequence shown in SEQ ID NO: 12.WSGR Docket No. 56146-747.601

[0074] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 80, a VH CDR2 having an amino acid sequence of SEQ ID NO: 81, a VH CDR3 having an amino acid sequence of SEQ ID NO: 82, VL CDR1 having an amino acid sequence of SEQ ID NO: 83, a VL CDR2 having an amino acid sequence of SEQ ID NO: 84, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 85. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0075] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 86, a VH CDR2 having an amino acid sequence of SEQ ID NO: 87, a VH CDR3 having an amino acid sequence of SEQ ID NO: 88, VL CDR1 having an amino acid sequence of SEQ ID NO: 89, a VL CDR2 having an amino acid sequence of SEQ ID NO: 90, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 91. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0076] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 92, a VH CDR2 having an amino acid sequence of SEQ ID NO: 93, a VH CDR3 having an amino acid sequence of SEQ ID NO: 94, VL CDR1 having an amino acid sequence of SEQ ID NO: 95, a VL CDR2 having an amino acid sequence of SEQ ID NO: 96, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 97. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0077] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 98, a VH CDR2 having an amino acid sequence of SEQ ID NO: 99, a VH CDR3 having an amino acid sequence of SEQ ID NO: 100, VL CDR1 having an amino acid sequence of SEQ ID NO: 89, a VL CDR2 having an amino acid sequence of SEQ ID NO: 102, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 103. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibodyWSGR Docket No. 56146-747.601(e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0078] In one instance, an anti-PD-1 binding domain comprises a VH CDR1 having an amino acid sequence of SEQ ID NO: 113, a VH CDR2 having an amino acid sequence of SEQ ID NO: 114, a VH CDR3 having an amino acid sequence of SEQ ID NO: 115, VL CDR1 having an amino acid sequence of SEQ ID NO: 83, a VL CDR2 having an amino acid sequence of SEQ ID NO: 117, and a VL CDR3 having an amino acid sequence of SEQ ID NO: 118. In some embodiments, the anti-PD-1 binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (i.e., SEQ ID NOs: 76 and 77, respectively) (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0079] In some embodiments, an immunocytokine composition comprises one of the antibodies described in Table 1A or IB below (optionally comprising one or more modifications to the Fc domain, hinge region, or other modification described herein) fused to a binding domain specific for VEGFA (e.g., any of the VEGFA binding domains described herein). In some embodiments, the binding domain specific for VEGFA is fused to the C-terminus of the heavy chain of the antibody described in Table 1A or IB. In some embodiments, the binding domain specific for VEGFA fused to the antibody of Table 1A or IB is an anti-VEGFA single-domain antibody as described herein.

[0080] In some embodiments, an anti-PD-1 binding domain comprises a single domain antibody. In some embodiments, the anti-PD-1 binding domain of a dual binding composition is a single domain antibody described in Table 1 A or IB, or a variant thereof.

[0081] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 2, a CDR2 as set forth in SEQ ID NO: 3, and a CDR3 as set forth in SEQ ID NO: 4. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 1 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 1 and retains the CDRs).

[0082] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 6, a CDR2 as set forth in SEQ ID NO: 7, and a CDR3 as set forth in SEQ ID NO: 8. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO:WSGR Docket No. 56146-747.6015 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 5 and retains the CDRs).

[0083] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 14, a CDR2 as set forth in SEQ ID NO: 15, and a CDR3 as set forth in SEQ ID NO: 16. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 13 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 13 and retains the CDRs).

[0084] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 18, a CDR2 as set forth in SEQ ID NO: 19, and a CDR3 as set forth in SEQ ID NO: 20. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 17 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 17 and retains the CDRs).

[0085] In some embodiments, the anti-PD-1 binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 288, a CDR2 as set forth in SEQ ID NO: 289, and a CDR3 as set forth in SEQ ID NO: 290. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-PD-1 binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 287 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 287 and retains the CDRs).

[0086] In some embodiments, an immunocytokine composition comprises one or two anti-PD-1 binding domains. In some embodiments, the immunocytokine composition comprises one anti- PD-1 binding domain. In some embodiments, the immunocytokine composition comprises two anti-PD-1 binding domains. In some embodiments, each anti-PD-1 binding domain is a Fab or scFv. In some embodiments, each anti-PD-1 binding domain is a single domain antibody. In some embodiments, the immunocytokine composition comprises two copies of the same anti-PD-1 binding domain. In some embodiments, the immunocytokine composition comprises two anti-PD- 1 Fabs. In some embodiments, the immunocytokine composition comprises one anti-PD-1 Fab and one anti-Pd-1 scFv. In some embodiments, the anti-PD-1 Fab and the anti-PD-1 scFv comprise the same VH and VL. In some embodiments, the immunocytokine composition comprises one anti-PD-1 Fab and one anti-PD-1 single domain antibody.WSGR Docket No. 56146-747.601TABLE 1A - Exemplary Antibodies Targeting PD-1 From Which anti-PD-1 binding domains can be derivedWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601Table IB - Additional PD-1 Binding DomainsWSGR Docket No. 56146-747.601

[0087] In some embodiments, the anti-PD-1 binding domain is one provided in Table 1C, or a derivative thereof. In some embodiments, the anti-PD-1 binding domain is one which comprises the CDRs of a VHH provided in Table 1C (e.g., those of VHH 47, 62, 70, 76, 84, or 178). In some embodiments, the anti-PD-1 binding domain is one which comprises the CDRs of a VHH provided in Table 1C and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding full-length VHH sequence (e.g., VHH 47, 62, 70, 76, 84, or 178). In some embodiments, the anti-PD-1 binding domain is one of the VHHs provided in Table 1C (e.g., VHH 47, 62, 70, 76, 84, or 178). In some embodiments, the andi-Pd- 1 binding domain is VHH47, or comprises the CDRs thereof.Table 1C - Exemplary anti-PD-1 VHH binding domainsWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601

[0088] In some embodiments, an anti-PD-1 VHH of the immunocytokine composition comprises one or more modifications which imparts the VHH with reduced immunogenicity or reduced binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acids Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP,WSGR Docket No. 56146-747.601KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., LI IK or LI IV) and / or Leu 89 (e.g., L89T)), and / . / / / etal., “A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944. doi.org / 10.1371 / journal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C- terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, the anti-PD-1 VHH (e.g., any of those described in the tables above) comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, the anti-PD-1 VHH comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two anti-PD-1 VHHs are linked in series (e.g., by a flexible peptide linker), in some instance only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C- terminal “SS” of each VHH).

[0089] In some embodiments, the anti-PD-1 binding domain of the immunocytokine composition comprises a light chain constant region (e.g., in cases in which the anti-PD-1 binding domain is a Fab). In some embodiments, the light chain constant region is one which contains one or more modifications which enhance the stability and / or manufacturability of the binding domain (or the immunocytokine composition as a whole). Such modifications are described in, for example, U.S. Patent No. 9,777,067 and U.S. Patent Publication No. US20150239977A1 and include, for example, modifications of residue LI 54 of the light chain constant region (EU numbering), such as an L154K substitution and / or modifications of residue L201 (EU numbering), such as an L201K substitution. In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises an L154K substitution compared to the consensus sequence (e.g., as in SEQ ID NO: 277). In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises an L201K substitution compared to the consensus sequence. In some embodiments, the light chain constant region of the anti-PD-1 binding domain (e.g., a Fab) comprises L154K and L201K substitutions compared to the consensus sequence (e.g., as in SEQ ID NO: 278).VEGFA Targeting Binding Domains

[0090] In some embodiments, an immunocytokine composition of the instant disclosure comprise one or more binding domains which target vascular endothelial growth factor A (VEGFA). In some embodiments, a binding domain incorporated into an immunocytokine composition of theWSGR Docket No. 56146-747.601 disclosure specifically binds to VEGFA. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with one or more of its receptors (e.g., VEGFR1, VEGFR2, and / or VEGFR3). In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with VEGFR1. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with VEGFR2. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with VEGFR3. In some embodiments, the anti- VEGFA binding domain is capable of disrupting and / or preventing the interaction of VEGFA with each of VEGFR1, VEGFR2, and VEGFR3.

[0091] A non-limiting, exemplary, human VEGFA amino acid sequence is LTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFVQL LGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGARKPG SWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSASRAGPG RASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRS YCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKP HQGQHIGEMSFLQHNKCECRCDKPRR (SEQ ID NO: 120) (UniProt ID A0A0A0MR43). Throughout the instant disclosure, “VEGF” and “VEGFA” are used interchangeably.

[0092] In some embodiments, the anti-VEGFA binding domain is comprised in an antigen binding fragment derived from an antibody. For example, the anti- VEGFA binding domain can be derived from any anti- VEGFA antibody known in the art or which can be made according to well understood methods. In some embodiments, the binding domain of an immunocytokine composition described herein is derived from an anti-VEGFA antibody or antigen binding fragment.

[0093] In one embodiment, an anti-VEGFA binding domain of an immunocytokine composition comprises and antigen binding fragment. In some embodiments, an anti-VEGFA binding domain of the disclosure comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) described herein, or those of an antibody or antigen binding fragment otherwise known in the art. In another embodiment, an anti- VEGFA binding domain of the disclosure comprises a combination of complementarity determining regions (VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3) described herein, orthose of an antibody or antigen binding fragment otherwise known in the art.

[0094] In one embodiment, an anti- VEGFA binding domain of the disclosure comprises the CDRs of an antibody selected from Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBB 05 incorporated into a VH and VL. In some embodiments, the anti- VEGFA binding domain comprises the VH and VL of any one of these antibodies. InWSGR Docket No. 56146-747.601 some embodiments, the anti-VEGFA binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0095] In some embodiments, an anti-VEGFA binding domain of the disclosure comprises the CDRs (e.g., VH and VL CDRs) of Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBI305 incorporated into a VH and VL. In one embodiment, an anti-PD-1 binding domain of the disclosure comprises the VH and VL of Bevacizumab, Brolucizumab, Faricimab, Ranibizumab, Ivonescimab, AI-081, HLX-04, or IBI305. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies in one of the following formats: a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, a bispecific diabody, triabody, a tetrabody, a maxibody, a Fab-Fc, a scFv-Fc, or a bispecific antibody. In some embodiments, the VH and VL of any one of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab, a Fab’, or an scFv. In some embodiments, the VH and VL any one of these antibodies or of the VH and VL which include the CDRs of these antibodies is comprised in a Fab or an scFv. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in a Fab. In some embodiments, the VH and VL of any of these antibodies or the VH and VL which include the CDRs of these antibodies is comprised in an scFv.

[0096] In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab, Brolucizumab, Faricimab, or Ranibizumab. In some embodiment, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab or Brolucizumab. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Bevacizumab. In some embodiments, the anti-VEGFA binding domain comprises the VH and VL of Brolucizumab.WSGR Docket No. 56146-747.601In some embodiments, the anti- VEGFA binding domain comprises the VH and VL of Bevacizumab in a Fab or scFv format.

[0097] TABLE 2A and 2B provide the sequences of exemplary anti-VEGFA antibodies and anti- VEGFA antigen binding fragments which contain sets of CDRs which can be incorporated into VHs and / or VLs and used as anti-VEGFA binding domains as described herein. In some embodiments, the VHs and VLs of the antibodies in Table 2 A or 2B are incorporated into anti- VEGFA binding domains (e.g., in a Fab or scFv format). In some embodiments, CDRs of a VH described in Table 2A or 2B is incorporated into a binding domain as a VHH. In some embodiments, a VH as described in Table 2 A or 2B is incorporated into a binding domain as a VHH.

[0098] In some instances, the sequences listed in Table 2A or 2B contain full-length heavy or light chains of the indicated antibodies with the VH or VL respectively indicated in bold. Where there is a reference herein to a VH or VL of a sequence in Table 2A or 2B which contains a full- length heavy or light chain, it is intended to reference the bolded portion of the sequence.

[0099] An anti-VEGFA binding domain can comprise a VH having an amino acid sequence of any one of those described in Table 2A or 2B. The anti- VEGFA binding domain can comprise (or further comprise) a VL having an amino acid sequence of any one of those described in Table 2 A or 2B. In preferred embodiments, the VH and VL are from the same antibody or antigen binding fragment described in Table 2A.

[0100] An anti- VEGFA binding domain can comprise a heavy chain, a VH, or a VH-CH1 domain (e.g., as in a Fab) having an amino acid sequence of any one of those set forth in Table 2A or 2B, or a portion corresponding to a VH thereof (e.g., the portion depicted in bold). An anti-VEGFA binding domain can comprise (or further comprise) a light chain (e.g., as in a Fab) or VL having an amino acid sequence of any one of those described in Table 2A or 2B, or a portion corresponding to a VL thereof. In preferred embodiments, the heavy chain, VH, or VH-CH1 domain and VL or light chain are from the same antibody or antigen binding fragment described in Table 2 A or 2B.

[0101] In some embodiments, an anti-VEGFA binding domain comprises a VH having an amino acid sequence shown in Table 2A, and a VL having an amino acid sequence shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Bevacizumab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Brolucizumab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Faricimab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Ranibizumab as shown in Table 2 A. InWSGR Docket No. 56146-747.601 some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Ivonescimab as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of AI-081 as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of HLX-04 as shown in Table 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of IBI305as shown in Table 2A.

[0102] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of an antibody or antigen binding fragment as shown in Table 2 A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of the same antibody or antigen binding fragment as shown in Table 2A comprised in a VL. In some embodiments, the anti- VEGFA binding domain comprises a corresponding VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of the parent antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0103] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Bevacizumab as shown in Table 2A comprising in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Bevacizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti- VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Bevacizumab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0104] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Brolucizumab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Brolucizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Brolucizumab as shown in Table 2 A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0105] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Faricimab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Faricimab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Faricimab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).WSGR Docket No. 56146-747.601

[0106] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Ranibizumab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Ranibizumab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Ranibizumab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0107] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of Ivonescimab as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of Ivonescimab as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of Ivonescimab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0108] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of AI-081 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of AI-081 as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of AI-081 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0109] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of HLX-04 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of HLX-04 as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of HLX-04 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0110] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of IB 1305 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of IBI305 as shown in Table 2A comprised in a VL. In some embodiments, the anti-VEGFA binding domain comprises a VH and VL having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100 % identity to the VH and VL of IBI305 as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).WSGR Docket No. 56146-747.601

[0111] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 202, a CDR2 as set forth in SEQ ID NO: 203, and a CDR3 as set forth in SEQ ID NO: 204. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 201 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 201 and retains the CDRs).

[0112] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 206, a CDR2 as set forth in SEQ ID NO: 207, and a CDR3 as set forth in SEQ ID NO: 208. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 205 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 205 and retains the CDRs).

[0113] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 210, a CDR2 as set forth in SEQ ID NO: 211, and a CDR3 as set forth in SEQ ID NO: 212. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 209 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 209 and retains the CDRs).

[0114] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 202, a CDR2 as set forth in SEQ ID NO: 215, and a CDR3 as set forth in SEQ ID NO: 216. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 213 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 213 and retains the CDRs).

[0115] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 218, a CDR2 as set forth in SEQ ID NO: 219, and a CDR3 as set forth in SEQ ID NO: 220. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 200, 217, 221, 280, or 281 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 200, 217, 221, 280, or 281 and retains the CDRs).WSGR Docket No. 56146-747.601

[0116] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 218, a CDR2 as set forth in SEQ ID NO: 279, and a CDR3 as set forth in SEQ ID NO: 220. In some embodiments, the CDRs are comprised in a VHH. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 282-284 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 282-284 and retains the CDRs).

[0117] In some embodiments, the anti-VEGFA binding domain is a single domain antibody comprising a CDR1 as set forth in SEQ ID NO: 223, a CDR2 as set forth in SEQ ID NO: 224, and a CDR3 as set forth in SEQ ID NO: 225. In some embodiments, the CDRs are comprised in a light chain single domain antibody. In some embodiments, the anti-VEGFA binding domain comprises an amino acid sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequence set forth in SEQ ID NO: 222 (e.g., the anti-VEGFA binding domain comprises the indicated sequence identity to SEQ ID NO: 222 and retains the CDRs).

[0118] In some embodiments, a dual binding composition comprises one of the antibodies described in Table 2A below (optionally comprising one or more modifications to the Fc domain, hinge region, or other modification described herein) fused to a binding domain specific for PD- 1 (e.g., any of the PD-1 binding domains described herein). In some embodiments, the binding domain specific for PD-1 is fused to the C-terminus of the heavy chain of the antibody described in Table 2 A. In some embodiments, the binding domain specific for PD-1 fused to the antibody of Table 2A is an anti-PD-1 single-domain antibody as described herein.

[0119] .In some embodiments, an immunocytokine composition comprises one or two anti- VEGFA binding domains. In some embodiments, the immunocytokine composition comprises one anti-VEGFA binding domain. In some embodiments, the immunocytokine composition comprises two anti-VEGFA binding domains. In some embodiments, each anti-VEGFA binding domain is a Fab or scFv. In some embodiments, each anti-VEGFA binding domain is a single domain antibody (e.g., a VHH). In some embodiments, the immunocytokine composition comprises two copies of the same anti-VEGFA binding domain. In some embodiments, the immunocytokine composition comprises two anti-VEGFA Fabs. In some embodiments, the immunocytokine composition comprises one anti-VEGFA Fab and one anti-VEGFA scFv. In some embodiments, the anti-VEGFA Fab and the anti-VEGFA scFv comprise the same VH and VL. In some embodiments, the immunocytokine composition comprises one anti-VEGFA Fab and one anti-VEGFA single domain antibody. In some embodiments, the immunocytokine composition comprises two anti-VEGFA single domain antibodies. In some embodiments, the immunocytokine composition comprises two anti-VEGFA VHHs.WSGR Docket No. 56146-747.601TABLE 2A - Exemplary Antibodies Targeting VEGFA From Which anti-VEGFA binding domains can be derivedWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601Table 2B - Single Domain anti-VEGFA AntibodiesWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601

[0120] In some embodiments, the anti-VEGFA binding domain is one provided in Table 2C, or a derivative thereof. In some embodiments, the anti-VEGFA binding domain is one which comprises the CDRs of a VHH provided in Table 2C. In some embodiments, the anti-VEGFA binding domain is one which comprises the CDRs of a VHH provided in Table 2C and comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding full-length VHH sequence. In some embodiments, the anti-VEGFA binding domain is one of the VHHs provided in Table 2C.Table 2C - Exemplary anti-VEGFA VHH binding domainsWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601

[0121] In some embodiments, the anti-VEGFA VHH of the immunocytokine composition comprises one or more modifications which imparts the VHH with reduced immunogenicity or reduced binding of pre-existing antibodies to the VHH. Examples of such modifications are described in, for example, U.S. Patent Publication Nos. US20180009888A9 (e.g., extension peptides of 1 to 5 extending beyond the C-terminal “SS” of the VHH, such as those consisting of the amino acids Ala and Gly), US20160207981A1 (e.g., substitutions of the C-terminal “SS” of the VHH, such as with an amino acid or peptide of a sequence E, SE, EG, SEG, EP, EPG, DP, DPG, K, SK, KP, KPG, RP, or RPG and / or substitutions of Leu 11 of the VHH, such as an LI IK, LI 1R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certain sequences from the VHH), US20170121399A1 (e.g., substitutions of Leu 11 of the VHH (e.g., LI IK or LI IV) and / or Leu 89 (e.g., L89T)), and Lin et al., “A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics,” PLoS ONE 16(7): e0254944.WSGR Docket No. 56146-747.601 doi.org / 10.1371 / journal.pone.0254944 (e.g., the addition of 1, 2, or 3 prolines beyond the C- terminal “SS” of the VHH, such as a two-proline peptide). In some embodiments, the anti-VEGFA VHH (e.g., any of those described in the tables above) comprises a C-terminal modification of the addition of 1, 2 or 3 prolines to the C-terminus of the VHH (e.g., to the C-terminus of any of the VHHs described herein). In some embodiments, the anti-VEGFA VHH comprises a C-terminal modification of the addition of 2 prolines to the C-terminus of the VHH. In embodiments where two anti-VEGFA VHHs are linked in series (e.g., by a flexible peptide linker), in some instance only the C-terminal VHH will comprise the modification (e.g., only the C-terminal VHH comprises the sequence “PP” after the C-terminal “SS” of the VHH). In some instances, both VHHs linked in series will contain the modification (e.g., both will comprise the sequence “PP” after the C-terminal “SS” of each VHH).

[0122] In some embodiments, the anti-VEGFA binding domain of the immunocytokine composition comprises a light chain constant region. In some embodiments, the light chain constant region is one which contains one or more modifications which enhance the stability and / or manufacturability of the binding domain (or the immunocytokine composition as a whole). Such modifications are described in, for example, U.S. Patent No. 9,777,067 and U.S. Patent Publication No. US20150239977A1 and include, for example, modifications of residue L154 of the light chain constant region (EU numbering), such as an L154K substitution and / or modifications of residue L201 (EU numbering), such as an L201K substitution. In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises an L154K substitution compared to the consensus sequence (e.g., as in SEQ ID NO: 277). In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises an L201K substitution compared to the consensus sequence. In some embodiments, the light chain constant region of the anti-VEGFA binding domain (e.g., a Fab) comprises L154K and L201K substitutions compared to the consensus sequence (e.g., as in SEQ ID NO: 278).

[0123] In some embodiments, an anti-VEGFA binding domain according to the instant disclosure can be a polypeptide which binds to VEGFA which is not derived from an antibody. In some embodiments, the anti-VEGFA binding domain comprises an anticalin which binds to VEGFA. In some embodiments, the anticalin comprises an amino acid sequence having at least 80%, 85%, 90% 95%, 96% 97%, 98%, 99%, or 100% identity to the sequenceDGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLKGHNLEAKVTMLISGRC QEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHGKPVRGVKLVGRD PKNNLEALEDFEKAAGRLSTESILIPRQSETCSPG (SEQ ID NO: 226). In some embodiments, the anti-VEGFA binding domain comprises the sequence of SEQ ID NO: 226.WSGR Docket No. 56146-747.601Cytokines

[0124] Cytokines are proteins produced in the body that are important in cell signaling. Cytokines can modulate the immune system, and cytokine therapy utilizes the immunomodulatory properties of the molecules to enhance or regulate the immune system of a subject. Disclosed herein in some embodiments are immunocytokines compositions which comprise cytokines (e.g., modified cytokines and / or synthetic cytokines) linked in immunocytokine compositions which comprise anti-PD-1 and anti-VEGFA binding domains. In some embodiments, immunocytokine compositions of the instant disclosure can exhibit enhanced biological activity compared to individual cytokines by themselves or can modulate the immune system in advantageous ways difficult to achieve with individual cytokines.

[0125] A cytokine of an immunocytokine composition as provided herein can be any cytokine. Non-limiting examples of cytokines include interleukins (e.g., IL-2, IL- 18, IL-7, IL- 17), TNF family cytokines e.g., TNF a, CD70, TNFSF14), interferons (e.g, IFNy, IFNa. IFN0), TGF-0 family cytokines (e.g., TGFB1, TGFB2, TGFB3), chemokines (e.g., CCL2, CCL3, CXCL9, CXCL10) and others. In some embodiments, the cytokine of the immunocytokine composition is an interleukin. In some embodiments, the interleukin is selected from an IL-1 family cytokine (e.g, IL-18, IL-ip, IL-33), an IL-2 family cytokine (e.g, IL-2, IL-4, IL-7, IL-15, IL-21), an IL-6 family interleukin (e.g., IL-6, IL-11, IL-31), an IL-10 family cytokine (e.g., IL-10, IL-19, IL-20, IL-22), an IL- 12 family cytokine (e.g., IL- 12, IL-23, IL-27, IL-35) and an IL- 17 family cytokine (e.g., IL-17, IL-17F, IL-25). In some embodiments, the cytokine of the immunocytokine composition is selected from an IL-2 polypeptide, an IL-7 polypeptide, an IL-12 polypeptide, and an IL- 18 polypeptide.

[0126] Cytokines of the immunocytokine compositions provided herein may be modified versions of the cytokines. In some embodiments, the cytokines comprise modifications (e.g., amino acid substitutions, additions, or deletions, attachment of polymers) which can modulate the activity of the cytokine (e.g., enhance activity, detune activity, or modulate the activity, such as by biasing the cytokine to one receptor or receptor subunit). In some embodiments, the cytokines comprise modifications in order to facilitate site specific attachment of a linker as provided herein. Additionally, cytokines provided herein may also be fused to additional polypeptides (e.g., antibody fusions, Fc fusions, etc.) or peptide sequences, such as artificial leader sequences, halflife extension sequences, or other peptides affixed to the N or C terminus of the cytokine. Cytokines may also be truncated versions of cytokines provided herein. In some embodiments, the cytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a naturally occurring cytokine (e.g., a human cytokine).IL-18 PolypeptidesWSGR Docket No. 56146-747.601

[0127] The present disclosure describes in some embodiments anti-VEGFA binding domains and / or anti-PD-1 binding domains linked to interleukin- 18 (IL- 18) polypeptides as immunocytokine compositions and their use as human therapeutic agents. IL-18 is a pro- inflammatory cytokine that elicits biological activities that initiate or promote host defense and inflammation following infection or injury. IL- 18 has been implicated in autoimmune diseases, myocardial function, emphysema, metabolic syndromes, psoriasis, inflammatory bowel disease, hemophagocytic syndromes, macrophage activation syndrome, sepsis, and acute kidney injury. In some models of disease, IL- 18 plays a protective role.

[0128] IL-18 also plays a major role in the production of IFNy from T-cells and natural killer cells. fFNy is a T helper type 1 cytokine mainly produced by T cells, NK cells, and macrophages and is critical for innate and adaptive immunity against viral, some bacterial, and protozoal infections. fFNy is also an important activator of macrophages and inducer of Class II major histocompatibility complex (MHC) molecule expression.

[0129] IL- 18 forms a signaling complex by binding to the IL- 18 alpha chain (IL-18Ra), which is the ligand binding chain for mature IL-18. However, the binding affinity of IL-18 to IL-18Ra is low. In cells that express the co-receptor, IL-18 receptor beta chain (IL-18RP), a high affinity heterodimer complex is formed, which then activates cell signaling.

[0130] The activity of IL-18 is balanced by the presence of a high affinity, naturally occurring IL- 18 binding protein (IL-18BP). IL-18BP binds IL-18 and neutralizes the biological activity of IL- 18. Cell surface IL-18Ra competes with IL-18BP for IL-18 binding. Increased disease severity can be associated with an imbalance of IL- 18 to IL-18BP such that levels of free IL-18 are elevated in the circulation. IL- 18 induces IFNy production, which in turn induces IL-18BP production. IL- 18BP then competes with IL-18Ra to inhibit IL-18 activity.

[0131] In some embodiments, the IL-18 polypeptides of the immunocytokines provided herein display reduced binding to IL-18BP and retain binding to the IL-18 receptor. The IL-18 polypeptides with this property provided herein are able to retain IL-18 receptor signaling activity (including inducing production of IFNy) even in the presence of IL-18BP. This allows the immunocytokines provided herein to retain IL- 18 signaling activity well beyond a short period of time after administration, or upon repeat administrations. In some embodiments, the IL- 18 polypeptides with this property comprise a modification (e.g., substitution, polymer attachment, or deletion) at one or more amino acid residues which convey this property to the IL-18 polypeptide. Examples of IL-18 polypeptides with this property are provided herein, as well as those otherwise known, such as those described in Patent Cooperation Treaty Publication No. W02019051015A1, WO2022038417A2 (corresponding U.S. Pat. Pub. No. US20220056091A1), and WO2023161856A1 (corresponding U.S. Pat. Pub. No. US20230357342A1), each of which isWSGR Docket No. 56146-747.601 hereby incorporated by reference as if set forth herein in its entirety. In addition to IL-18 polypeptides provided herein, these otherwise known IL- 18 polypeptides or their analogs may similarly be modified with points of attachment to the linker as provided herein. In some embodiments, an IL-18 can by prepared synthetically (e.g., via chemical synthesis).

[0132] In some embodiments, it is desirable to use a “detuned” IL- 18 polypeptide in an immunocytokine composition. Use of a detuned IL-18 polypeptide may exhibit fewer toxic effects in vivo upon administration. However, due to the targeting of the IL- 18 polypeptide to a desired area due to the affinity of the binding domains for their targets, the IL-18 polypeptide can still be sufficiently active locally. Such a “detuned” IL- 18 polypeptide is one which is less potent in signaling through the IL- 18 receptor (e.g., has a higher EC50 for IFNg induction) than wild type IL-18. In some embodiments, such a “detuned” IL- 18 polypeptide exhibits at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold lower potency than WT IL- 18. In some embodiments, the detuned IL- 18 polypeptide exhibits a potency which is between 10-fold and 1000-fold lower than WT IL- 18, between 50-fold and 500- fold lower than WT IL- 18, between 100-fold and 1000-fold lower than WT IL- 18, or another range.Points of Attachment of Linkers to IL-18 Polypeptides Attached to the Immunocytokine Compositions for Conjugation

[0133] In some embodiments, the immunocytokines provided herein comprise linkers which have a point of attachment to the IL- 18 polypeptide. In some embodiments, the linker is a chemical linker. The linker has another point of attachment to the immunocytokine composition (e.g., to an Fc domain of the immunocytokine composition) as described herein elsewhere (e.g., a K248 residue of an Fc domain (EU numbering)). The point of attachment to the IL-18 polypeptide is to a residue as provided herein below.

[0134] In some embodiments, the linker is attached to an amino acid residue of the IL-18 polypeptide. In some embodiments, the linker is attached to any amino acid residue of the IL-18 polypeptide (e.g., at a position corresponding to any one of positions 1-157 of SEQ ID NO: 701). In some embodiments, the linker is attached at a non-terminal residue of the IL- 18 polypeptide (e.g., a residue at position corresponding to any one of positions 2-156 of SEQ ID NO: 701). In some embodiments, the linker is attached at a non-terminal residue of the IL- 18 polypeptide, wherein the IL- 18 polypeptide has been extended or truncated by one or more amino acids relative to SEQ ID NO: 701.

[0135] In some embodiments, the linker is attached to the IL-18 polypeptide at a residue in a region comprising residues 2-156, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the linker is attached to the IL-18 polypeptideWSGR Docket No. 56146-747.601 at a residue in a region comprising residues 30-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue in a region comprising residues 33-43, residues 60-100, residues 65-75, residues 80-90, residues 85-100, residues 90-110, residues 115-130, residues 120- 130, or residues 140-150. In some embodiments, the linker is attached to the IL-18 polypeptide at a residue selected from residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, and 144. In some embodiments, the linker is attached to the IL- 18 polypeptide at a residue selected from 68, 69, 70,85, 86, and 98. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 68, 69, or 70. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 85,86, 95, or 98. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 68. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 69. In some embodiments, the linker is attached to the IL-18 polypeptide at residue 70. In some embodiments, the linker is attached to the IL- 18 polypeptide at residue 85. In some embodiments, the linker is attached to the IL- 18 polypeptide at residue 86. In some embodiments, the linker is attached to the IL- 18 polypeptide at residue 95. In some embodiments, the linker is attached to the IL- 18 polypeptide at residue 98.

[0136] In some embodiments, the linker is attached to the IL- 18 polypeptide at a residue which is known in the art to be compatible with attachment of a polymer to the IL-18 polypeptide without having a profound impact on the bioactivity of the IL-18 polypeptide. Examples of these residues include residues 38, 76, 78, 121, 127, and 144, as described in PCT Pub. No. W02004091517A2, which is hereby incorporated by reference as if set forth in its entirety.

[0137] In some embodiments, the residue to which the linker is attached is a natural amino acid residue. In some embodiments, the residue to which the linker is covalently attached is selected from cysteine, aspartate, asparagine, glutamate, glutamine, serine, threonine, lysine, and tyrosine. In some embodiments, the residue to which the linker is covalently attached is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the linker is covalently attached to a cysteine. In some embodiments, the linker is covalently attached to a lysine. In some embodiments, the linker is covalently attached to a glutamine. In some embodiments, the linker is covalently attached to an asparagine. In some embodiments, the residue to which the linker is attached is a tyrosine. In some embodiments, the residue to which the linker is attached is the natural amino acid in that position in SEQ ID NO: 701.

[0138] In some embodiments, the linker is attached to a different natural amino acid which is substituted at the relevant position. The substitution can be for a naturally occurring amino acid which is more amenable to attachment of additional functional groups (e.g., aspartic acid, cysteine, glutamic acid, lysine, serine, threonine, or tyrosine), a derivative of modified version of anyWSGR Docket No. 56146-747.601 naturally occurring amino acid, or any unnatural amino acid e.g., an amino acid containing a desired conjugation handle, such as a CLICK chemistry reagent such as an azide, alkyne, etc.). In some embodiments, the linker is covalently attached to site-specifically to a natural amino acid.

[0139] In some embodiments, the linker is attached at an unnatural amino acid residue. In some embodiments, the unnatural amino acid residue comprises a conjugation handle. In some embodiments, the conjugation handle facilitates the addition of the linker to the IL- 18 polypeptide. The conjugation handle can be any of the conjugation handles provided herein. In some embodiments, the linker is covalently attached site-specifically to the unnatural amino acid. Nonlimiting examples of amino acid residues comprising conjugation handles can be found, for example, in PCT Pub. Nos. WO2015054658A1, WO2014036492 Al, WO2021133839A1 W02006069246A2, and W02007079130A2, each of which is incorporated by reference as if set forth in its entirety.

[0140] In some embodiments, the linker is covalently attached at residue 68. In some embodiments, the linker is covalently attached at residue C68, C68E, C68D, C68Q, C68K, C68N, or C68Y. In some embodiments, the linker is covalently attached at residue C68. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 68. In preferred embodiments of the instant disclosure, the linker is attached to residue C68 of the IL-18 polypeptide (e.g., C68 of SEQ ID NO: 702, for example).

[0141] In some embodiments, the linker is covalently attached at residue 69. In some embodiments, the linker is covalently attached at residue E69, E69C, E69D, E69Q, E69K, E69N, or E69Y. In some embodiments, the linker is covalently attached at residue E69. In some embodiments, the linker is covalently attached residue E69C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 69.

[0142] In some embodiments, the linker is covalently attached at residue 70. In some embodiments, the linker is covalently attached at residue K70, K70C, K70D, K70Q, K70E, K70N, or K70Y. In some embodiments, the linker is covalently attached at residue K70. In some embodiments, the linker is covalently attached residue K70C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 70.

[0143] In some embodiments, the linker is covalently attached at residue 85. In some embodiments, the linker is covalently attached at residue E85, E85C, E85D, E85Q, E85K, E85N, or E85Y. In some embodiments, the linker is covalently attached at residue E85. In some embodiments, the linker is covalently attached residue E85C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 85.

[0144] In some embodiments, the linker is covalently attached at residue 86. In some embodiments, the linker is covalently attached at residue M86C, M86D, M86Q, M86K, M86N,WSGR Docket No. 56146-747.601M86E, or M86Y. In some embodiments, the linker is covalently attached M86C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 86.

[0145] In some embodiments, the polymer is covalently attached at residue 95. In some embodiments, the polymer is covalently attached at residue T95, T95C, T95D, T95Q, T95K, T95N, T95E, or T95 Y. In some embodiments, the polymer is covalently attached at residue T95C, T95D, T95Q, T95K, T95N, T95E, or T95Y. In some embodiments, the polymer is covalently attached at residue T95C. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 95.

[0146] In some embodiments, the linker is covalently attached at residue 98. In some embodiments, the linker is covalently attached at residue D98, D98C, D98Q, D98K, D98N, D98E, or D98Y. In some embodiments, the linker is covalently attached at residue D98C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 98.

[0147] In some embodiments, the linker is covalently attached through a modified natural amino acid. In some embodiments, the modified natural amino acid comprises a conjugation handle. In some embodiments, the linker is covalently attached through a modified amino acid a. In some embodiments, the modified amino acid a is an amino-acid-PEG-azide group. In some embodiments, the modified amino acid a is a glutamate, aspartate, lysine, cysteine, or tyrosine modified to incorporate an azide group linked to the amino acid through a PEG spacer. In some embodiments, the modified amino acid a has a structure selected from:wherein each n is independently an integer from 1-30. In some embodiments, n is an integer from 1-20, 1-10, 2-30, 2-20, 2-10, 5-30, 5-20, or 5-10. In some embodiments, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 6. In some embodiments, n is 12. The modified amino acid a can be incorporated at any point of attachmentWSGR Docket No. 56146-747.601 of the IL- 18 polypeptide as provided herein. In some embodiments, the modified amino acid a is located at a position on the IL- 18 polypeptide selected from residue 68, residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.

[0148] Where IL- 18 polypeptides contain unnatural amino acids or modified natural amino acids (e.g., those provided herein for purposes of conjugation), these amino acids may be incorporated into the IL- 18 polypeptides using many techniques known in the art for introduction such modifications. For example, recombinant proteins with unnatural amino acids can be made using methods as described in Patent Cooperation Treaty Publication Nos. WO2016115168, W02002085923, W02005019415, and W02005003294. Alternatively or in combination, unnatural or modified natural amino acids can be incorporated into chemically synthesized proteins during synthesis.

[0149] In some embodiments, the linker is attached to the C-terminus of the IL- 18 polypeptide, optionally by a linking peptide (e.g., any one of the linking peptides described herein). In some embodiments, the linker is attached using a sortase mediated reaction. For example, an IL-18 polypeptide described herein can have a sortase recognition sequence (e.g., LPETGGH (SEQ ID NO: 713)) appended to the C-terminus of the IL-18 polypeptide, optionally via a linking peptide, such as those described herein. The IL-18 polypeptide (e.g., as incorporated into an Act-IL-18 polypeptide) can then be reacted with a suitable donor group (e.g., an additional group containing an N-terminal glycine, preferably a polyglycine). The donor group can be a polypeptide (e.g., a full length protein desired to be attached to the Act-IL-18 polypeptide), or can be a small peptide comprising a conjugation handle (e.g., a short peptide of, e.g., 2-10 amino acids, which comprises at least 1, preferably 2-3 N-terminal glycine residue(s) linked to a suitable conjugation handle). Exemplary linkers which can be attached via sortase include the GGK-PEG9-N3 and GGE-PEG9- N3 reagents shown below, though other similar such linkers can also be added (e.g., ones which have variable PEG sizes, alternative amino acids attached to the GG group, alternative conjugation handles, etc.). As is apparent to one of ordinary skill in the art, in the sortase region, the “GGH” portion of the sortase recognition sequence is replaced by the glycine (or polyglycine) containing sequence of the reagent (or additional group polypeptide as the case may be). Descriptions of sortase mediated conjugation to proteins can be found in, for example, Patent Cooperation Treaty Publication Nos. W02013 / 003555A1, WO2014 / 140317A2, and W02020 / 007899A1.GGE-PEG9-N3WSGR Docket No. 56146-747.601GGK-PEG9-N3Modifications to IL-18 polypeptides

[0150] In some embodiments, the IL-18 polypeptide of the immunocytokine comprises one or more modifications to that of SEQ ID NO: 701. The modifications provided herein are in addition to any modification at the point of attachment as discussed supra. In some embodiments, the residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL- 18 polypeptide of the immunocytokine composition can be wild type IL-18 (i.e., SEQ ID NO: 701).

[0151] Modifications to the IL-18 polypeptide described herein encompass mutations, addition of various functionalities, deletion of amino acids, addition of amino acids, or any other alteration of the wild-type version of the protein or protein fragment. Functionalities which may be added to polypeptides include polymers, linkers, alkyl groups, detectable molecules such as chromophores or fluorophores, reactive functional groups, or any combination thereof. In some embodiments, functionalities are added to individual amino acids of the polypeptides. In some embodiments, functionalities are added site-specifically to the polypeptides.

[0152] In some embodiments, the IL- 18 polypeptide of the immunocytokine comprise one or more modifications in addition to a modification needed to attach the linker to the relevant residue of the IL-18 polypeptide (e.g., an amino acid substitution at a residue to which the linker is not attached). In some embodiments, the modification is in the range of amino acid residues 1-127, based on the sequence of human IL-1837'193(SEQ ID NO: 701). SEQ ID NO: 701 reflects the bioactive form of IL-18. Endogenously, IL-18 is initially expressed with an additional 36 amino acid segment at the N-terminus which is cleaved by caspases to mediate biologic activity.

[0153] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more modified amino acid residues.

[0154] In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine exhibits an EC50 value for fFNy release in parental NK92 cells of at most about 10 nM, at most about 5 nM, at most about 2 nM, at most about 1 nM, at most about 0.5 nM, at most about 0.25WSGR Docket No. 56146-747.601 nM, or at most about 0.1 nM. In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine exhibits an EC50 value for IFNy release in parental NK92 cells of at most least 0.001 nM, 0.005 nM, 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, or 0.05 nM. In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine exhibits an EC 50 value for IFNy release in parental NK92 cells of 0.001 nM to 5 nM, 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.001 nM to 2 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.001 nM to 1 nM, 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.001 nM to 0.5 nM, 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.001 nM to 0.25 nM, 0.01 nM to 0.25 nM, 0.05 nM to 0.25 nM, 0.001 nM to 0.1 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM.

[0155] In some embodiments, the IL- 18 polypeptide incorporated into the immunocytokine as in the format depicted in FIG. 6B exhibits an EC50 value for IFNy release in parental NK92 cells of at most about 10 nM, at most about 5 nM, at most about 2 nM, at most about 1 nM, at most about 0.5 nM, at most about 0.25 nM, or at most about 0.1 nM. In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine of format FIG. 6B exhibits an EC50 value for IFNy release in parental NK92 cells of at most least 0.001 nM, 0.005 nM, 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, or 0.05 nM. In some embodiments, the IL-18 polypeptide incorporated into the immunocytokine of format FIG. 6B exhibits an EC50 value for IFNy release in parental NK92 cells of 0.001 nM to 5 nM, 0.01 nM to 5 nM, 0.05 nM to 5 nM, 0.001 nM to 2 nM, 0.01 nM to 2 nM, 0.05 nM to 2 nM, 0.001 nM to 1 nM, 0.01 nM to 1 nM, 0.05 nM to 1 nM, 0.001 nM to 0.5 nM, 0.01 nM to 0.5 nM, 0.05 nM to 0.5 nM, 0.001 nM to 0.25 nM, 0.01 nM to 0.25 nM, 0.05 nM to 0.25 nM, 0.001 nM to 0.1 nM, 0.01 nM to 0.1 nM, 0.05 nM to 0.1 nM.

[0156] In some embodiments, the IL- 18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 701. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 702. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 703. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 704. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 705. In some embodiments, the IL- 18 polypeptideWSGR Docket No. 56146-747.601 of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 706. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 707. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 708. In some embodiments, the IL- 18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 709. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 710. In some embodiments, the IL-18 polypeptide of the immunocytokine comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 711.

[0157] In some embodiments, the IL- 18 polypeptide of the immunocytokine provided herein comprises an amino acid sequence of any one of SEQ ID NOs: 702-711 provided herein. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of any one of SEQ ID NOs: 702-711. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 702. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 702. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence of SEQ ID NO: 703. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 703.

[0158] In some embodiments, the IL-18 polypeptide of the immunocytokine described herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 9 amino acid substitutions, wherein the amino acid substitutions are relative to SEQ ID NO: 701. In some embodiments, the IL- 18 polypeptide comprises 1 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 1 or 2 amino acid substitutions, 1 to 3 amino acid substitutions, 1 to 4 amino acid substitutions, 1 to 5 amino acid substitutions, 1 to 6 amino acid substitutions, 1 to 7 amino acid substitutions, 1 to 8 amino acid substitutions, 2 to 3 amino acid substitutions, 2 to 4 amino acid substitutions, 2 to 5 amino acid substitutions, 2 to 6 amino acid substitutions, 2 to 7 amino acid substitutions, 2 to 8 amino acid substitutions, 2 to 9 amino acid substitutions 3 or 4 amino acid substitutions, 3 to 5 amino acid substitutions, 3 to 6 amino acidWSGR Docket No. 56146-747.601 substitutions, 3 to 7 amino acid substitutions, 3 to 9 amino acid substitutions, 4 or 5 amino acid substitutions, 4 to 6 amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 amino acid substitutions, 5 or 6 amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 amino acid substitutions, 6 or 7 amino acid substitutions, 6 to 9 amino acid substitutions, or 7 to 9 amino acid substitutions. In some embodiments, the IL-18 polypeptide comprises 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions. In some embodiments, the IL- 18 polypeptide comprises at most 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, or 9 amino acid substitutions.

[0159] In some embodiments, one modification is at amino acid residue 6. In some embodiments, one modification is in the range of amino acid residues 53-63. In some embodiments, one modification is at amino acid residue 53. In some embodiments, one modification is at amino acid residue 63.

[0160] In some embodiments, the IL-18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 701 selected from: Y01X, F02X, E06X, S10X, VI IX, D17X, C38X, M51X, K53X, D54X, S55X, T63X, C68X, C76X, AND C127X, wherein each X is independently a natural or non-natural amino acid. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98. In some embodiments, the IL- 18 polypeptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 701 selected from: Y01G, F02A, E06K, S10T, VI II, D17N, C38S, C38A, C38Q, M51G, K53A, D54A, S55A, T63A, C68S, C68A, C76S, C76A, C127A, and C127S. In some embodiments, the IL- 18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as E69C, K70C, E85C, M86C, T95C, or D98C.

[0161] In some embodiments, the interleukin- 18 (IL-18) polypeptide comprising E06K and K53A, wherein residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-18 polypeptide further comprises VI II. In some embodiments, the IL- 18 polypeptide further comprises T63A. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01X, S55X, F02X, D54X, C38X, C68X, E69X, K70X, C76X, or C127X, wherein each X is independently an amino acid or an amino acid derivative. In some embodiments, the IL-18 polypeptide further comprises at least one of Y01G, S55A, F02A, D54A, C38S, C38A, C38Q, C68S, C68A, E69C, K70C, C76S, C76A, C127S, or C127A. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at the point of attachment of the linker, such as residue 69, residue 70, residue 85, residue 86, residue 95, or residue 98.WSGR Docket No. 56146-747.601

[0162] In some embodiments, the IL-18 peptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 701, wherein the modification is E06X, VI IX, K53X, S55X, or T63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL-18 peptide comprises at least two modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06X and K53X; E06X and S55X; K53X and S55X; E06X and T63X; or K53X and T63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL- 18 peptide comprises at least three modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06X, K53X, and S55X; or E06X, K53X, and T63X, wherein X is a natural or non-natural amino acid. In some embodiments, the IL-18 peptide comprises at least four modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06X, K53X, S55X, and T63X; E06X, K53X, S55X, and Y01X; E06X, K53X, S55X, and F02X; E06X, K53X, S55X, and D54X; E06X, K53X, S55X, and M51X; or C38X, C68X, C76X, and C127X, wherein X is a natural or non-natural amino acid. In each embodiment wherein a plurality of amino acids residues are replaced with a natural or non-natural amino acid X, each X is independently the same or a different amino acid.

[0163] In certain embodiments, the IL-18 polypeptide comprises a substitution at residue Yl. In certain embodiments, the IL-18 polypeptide can comprise YIM substitution. Unless specifically mentioned otherwise, the residue position numbering is provided in this paragraph, and elsewhere in this disclosure is based on SEQ ID NO: 701, as a reference sequence. Unless specifically mentioned otherwise, the amino acid substitutions provided in this paragraph, and elsewhere in this disclosure is with respect to SEQ ID NO: 701, as a reference sequence. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue F2. In certain embodiments, the IL-18 polypeptide can comprise F2A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue E6. In certain embodiments, the IL-18 polypeptide comprises E6K substitution. In certain embodiments, the IL- 18 polypeptide comprises E6R substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue K8. In certain embodiments, the IL-18 polypeptide comprises K8L substitution. In certain embodiments, the IL- 18 polypeptide comprises K8E substitution. In certain embodiments, the IL- 18 polypeptide comprises K8R substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue VI 1. In certain embodiments, the IL-18 polypeptide can comprise VI II substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue E31. In certain embodiments, the IL- 18 polypeptide comprises E31A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue T34. In certain embodiments, the IL-18 polypeptide comprises T34A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D35. In certainWSGR Docket No. 56146-747.601 embodiments, the IL-18 polypeptide comprises D35A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue S36. In certain embodiments, the IL-18 polypeptide comprises S36A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D37. In certain embodiments, the IL-18 polypeptide comprises D37A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D40. In certain embodiments, the IL- 18 polypeptide comprises D40A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue N41. In certain embodiments, the IL-18 polypeptide comprises N41A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue 149. In certain embodiments, the IL-18 polypeptide comprises I49E substitution. In certain embodiments, the IL-18 polypeptide comprises I49M substitution. In certain embodiments, the IL-18 polypeptide comprises I49R substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue K53. In certain embodiments, the IL- 18 polypeptide comprises K53A substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue D54. In certain embodiments, the IL-18 polypeptide comprises D54A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue S55. In certain embodiments, the IL-18 polypeptide comprises S55A substitution. In certain embodiments, the IL-18 polypeptide comprises S55T substitution. In certain embodiments, the IL-18 polypeptide comprises S55H substitution. In certain embodiments, the IL-18 polypeptide comprises S55R substitution. In certain embodiments, the IL- 18 polypeptide comprises a substitution at residue T63. In certain embodiments, the IL-18 polypeptide comprises T63A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue Q103. In certain embodiments, the IL-18 polypeptide comprises Q103R substitution. In certain embodiments, the IL-18 polypeptide comprises Q103E substitution. In certain embodiments, the IL- 18 polypeptide comprises Q103K substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue G108. In certain embodiments, the IL-18 polypeptide comprises G108A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue H109. In certain embodiments, the IL-18 polypeptide comprises H109A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue DUO. In certain embodiments, the IL-18 polypeptide comprises D110A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue D132. In certain embodiments, the IL-18 polypeptide comprises D132A substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue VI 53. In certain embodiments, the IL-18 polypeptide comprises V153R substitution. In certain embodiments, the IL- 18 polypeptide comprises V153E substitution. In certain embodiments, the IL- 18 polypeptide comprises V153Y substitution. In certainWSGR Docket No. 56146-747.601 embodiments, the IL-18 polypeptide comprises a substitution at residue C38. In certain embodiments, the IL- 18 polypeptide comprises C38A substitution. In certain embodiments, the IL-18 polypeptide comprises C38S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C68. In certain embodiments, the IL-18 polypeptide comprises C68A substitution. In certain embodiments, the IL-18 polypeptide comprises C68S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C76. In certain embodiments, the IL- 18 polypeptide comprises C76A substitution. In certain embodiments, the IL-18 polypeptide comprises C76S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C127. In certain embodiments, the IL-18 polypeptide comprises C127A substitution. In certain embodiments, the IL-18 polypeptide comprises C127S substitution. In certain embodiments, the IL-18 polypeptide comprises a substitution at residue C38, C68, C76, and / or C127. In certain embodiments, the IL-18 polypeptide comprises a C38A, C38S, C68A, C68S, C76A, C76S, C127A, and / or C127S substitution. In certain embodiments, the IL-18 polypeptide comprises C38A, C76A, and C127A substitutions. In certain embodiments, the IL-18 polypeptide comprises C38S, C76S and C127S substitutions. In certain embodiments, the IL-18 polypeptide comprises C38A, C68A, C76A, and C127A substitutions. In certain embodiments, the IL-18 polypeptide comprises C38S, C68S, C76S and C127S substitutions.

[0164] In some embodiments, the IL-18 peptide comprises at least one modification to the amino acid sequence of SEQ ID NO: 701, wherein the modification is E06K, VI II, K53A, S55A, or T63A. In some embodiments, the IL- 18 peptide comprises at least two modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06K and K53A; E06K and S55A; K53 A and S55A; E06K and T63 A; or K53 A and T63 A. In some embodiments, the IL- 18 peptide comprises at least three modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06K, K53A, and S55A; E06K, VI II, and K53A; E06K, C38A, and K53A; or E06K, K53A, and T63A. In some embodiments, the IL- 18 peptide comprises at least four modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06K, K53A, S55A, and T63A; E06K, K53A, S55A, and Y01G; E06K, K53A, S55A, and F02A; E06K, K53A, S55A, and D54A; E06K, K53A, S55A, and M51G; or C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 peptide comprises at least six modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modifications comprise E06K, K53 A, C38S, C68S, C76S, and C127S; or K53 A, T63 A, C38S, C68S, C76S, and C127S. In some embodiments, the IL-18 polypeptide comprises at least seven modifications to the sequence of SEQ ID NO: 701, wherein the seven modifications comprise E6K, VI II, C38A, K53A, T63A, C76A, C127A. In some embodiments, the IL-18 peptide comprises at least eight modifications to the amino acid sequence of SEQ ID NO: 701, wherein the modificationsWSGR Docket No. 56146-747.601 comprise Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A. In some embodiments, the IL-18 peptide comprises at least eight modifications to the amino acid sequence of SEQ ID NO:701, wherein the modifications comprise Y01G, F02A, E06K, M51G, K53A, D54A, S55A, and T63A.

[0165] In some embodiments, IL- 18 polypeptide as provided herein comprises E06K and K53A, wherein residue position numbering of the IL-18 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 702. In some embodiments, the IL-18 polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identical to the amino acid sequence of SEQ ID NO: 703. In some embodiments, the IL-18 polypeptide further comprises an amino acid substitution at one or more cysteine residues. In some embodiments, the IL-18 polypeptide comprises one or more cysteines substituted with either serine or alanine. In some embodiments, the IL-18 polypeptide comprises amino acid substitutions at each cysteine residue of SEQ ID NO: 701.

[0166] In some embodiments, the IL- 18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100 % sequence identity to SEQ ID NO: 702-712. In some embodiments, the IL- 18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100 % sequence identity to SEQ ID NO: 703. In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity to SEQ ID NO: 704. In some embodiments, the IL-18 polypeptide comprises a polypeptide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99 % sequence identity to SEQ ID NO: 705. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 706. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO:702. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO: 702. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO: 702. In some embodiments, the polypeptide sequence is at least about 98% identical to SEQ ID NO: 702. In some embodiments, the polypeptide sequence is identical to SEQ ID NO: 702. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 703. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO: 703. In some embodiments, the polypeptide sequence is at least about 95% identical toWSGR Docket No. 56146-747.601SEQ ID NO: 703. In some embodiments, the polypeptide sequence is at least about 98% identical to SEQ ID NO: 703. In some embodiments, the polypeptide sequence is identical to SEQ ID NO:703. In some embodiments, the polypeptide sequence is at least about 80% identical to SEQ ID NO: 704. In some embodiments, the polypeptide sequence is at least about 90% identical to SEQ ID NO: 704. In some embodiments, the polypeptide sequence is at least about 95% identical to SEQ ID NO: 704. In some embodiments, the polypeptide sequence is at least about 98% identical to SEQ ID NO: 704. In some embodiments, the polypeptide sequence is identical to SEQ ID NO:704. In some embodiments, the IL- 18 polypeptide is recombinant. In some embodiments, the IL- 18 polypeptide is one provided in the Tables below listing IL- 18 variants.Activatable IL- 18 Polypeptides

[0167] In some embodiments, an IL- 18 polypeptide incorporated into an immunocytokine composition of the disclosure is an activatable IL- 18 polypeptide (Act-IL-18 polypeptide). Nonlimiting examples of activatable IL- 18 polypeptide compatible with the instant disclosure are described in, for example, WO2023161853A1 (corresponding US Pat. Pub. No. US20240116997A1), the contents of which are incorporated by reference as if set forth herein in its entirety.

[0168] In some embodiments, an Act-IL-18 polypeptides of the disclosure comprises an artificial polypeptide attached to an IL- 18 polypeptide. The artificial polypeptide comprises a cleavable group which, when cleaved, releases all or a portion of the artificial polypeptide. Upon cleavage, the Act-IL-18 is converted into an active form of the IL-18 polypeptide which is capable of performing IL-18 activity. In some embodiments, the artificial polypeptide inhibits the Act-IL-18 polypeptide from interacting with and / or signaling through an IL- 18 receptor. In some embodiments, the artificial polypeptide is capable of undergoing a change in response to a condition or stimulus which results in a conversion of the Act-IL-18 polypeptide into an active IL-18 polypeptide. In some embodiments, the change is a cleavage of at least a portion of the artificial polypeptide from the IL- 18 polypeptide.

[0169] In some embodiments, an activatable interleukin- 18 (Act-IL-18) polypeptide comprises an artificial polypeptide attached to an interleukin- 18 (IL-18) polypeptide (i.e., any of the IL-18 polypeptide described herein supra, wherein the artificial polypeptide comprises a protease cleavage site, and wherein cleavage at the protease cleavage site converts the Act-IL-18 into an active form of the IL-18 polypeptide.Artificial Polypeptides

[0170] In one aspect, provided herein, are artificial polypeptides attached to IL- 18 polypeptides of Act-IL-18 polypeptides. Artificial polypeptides as provided herein serve to detune the activity of the IL-18 polypeptide while they are attached in an intact form. In some embodiments, cleavageWSGR Docket No. 56146-747.601 of the artificial polypeptide serves to activate the IL-18 polypeptide (e.g., allowing the IL-18 polypeptide to signal through IL-18Rab). An artificial polypeptide provided herein can be attached to any residue. In some embodiments, the artificial polypeptide is an artificial polypeptide (e.g., attached to a terminal residue of the IL- 18 polypeptide).

[0171] In some embodiments, the artificial polypeptide can have other functionalities attached (e.g., in addition to being attached to the IL-18 polypeptide, the artificial polypeptide is also attached to another group, such as an additional polypeptide (e.g., antibody, dummy receptor, another cytokine), a half-life extension polymer (e.g., poly(ethylene glycol) (PEG)), or another desired functionality). In some embodiments, cleavage of the artificial polypeptide serves also to cleave this additional group from the IL- 18 polypeptide.

[0172] In one aspect, the Act-IL-18 polypeptides provided herein comprise an artificial polypeptide attached to a terminal residue of the IL- 18 polypeptide. In some embodiments, the artificial polypeptide is covalently attached to the IL-18 polypeptide. In some embodiments, the artificial polypeptide is a group which is not naturally attached to the terminus of a WT IL-18 polypeptide, such as the natural precursor 36 amino acid propeptide (or a variant thereof) attached the IL- 18 by a protease recognition sequence which is not present in the full-length, immature IL- 18 polypeptide (E.g., the caspase recognition sequence is swapped with a different recognition sequence). In some embodiments, the artificial polypeptide can be the natural propeptide, or a variant thereof as described elsewhere herein, preferably connected to the IL- 18 polypeptide via a cleavable linker sequence (i.e., a protease recognition sequence) as described herein (e.g., as set forth in Table 4 described herein). In some embodiments, the artificial polypeptide is engineered to possess the properties provided herein. In some embodiments, the artificial polypeptide is fused to an IL-18 polypeptide (e.g., as a fusion protein).

[0173] In some embodiments, an artificial polypeptide provided herein inhibits at least one activity associated with an IL- 18 polypeptide, such as the ability to bind to an IL- 18 receptor or effectuate signaling through the IL-18 receptor (IL-18Rab) (e.g., inducing production of I Ny in an immune cell). In some embodiments, when the artificial polypeptide is intact, the Act-IL-18 polypeptide is in an inactive state (e.g., lacks or has a substantially diminished ability to bind IL- 18Rab or signal through IL-18Rab).

[0174] In some embodiments, the presence of the intact artificial polypeptide on the IL-18 polypeptide results in the Act-IL-18 polypeptide displaying a binding affinity to IL-18Rab or an IL-18R subunit which is at least 10-fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000-fold lower than WT IL-18. In some embodiments, the presence of the intact artificial polypeptide on the IL- 18 polypeptide results in the Act-IL-18 polypeptide displaying a binding affinity to IL-18Rab or an IL-18R subunit which is at least 10-WSGR Docket No. 56146-747.601 fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000- fold lower than the IL- 18 polypeptide without the artificial polypeptide.

[0175] In some embodiments, the presence of the intact artificial polypeptide on the IL-18 polypeptide results in the Act-IL-18 polypeptide displaying an ability to induce fFNy production in a cell (e.g., an immune cell such as an NK cell) which is at least 10-fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000-fold lower than WT IL-18. In some embodiments, the presence of the intact artificial polypeptide on the IL-18 polypeptide results in the Act-IL-18 polypeptide displaying an ability to induce IFNy production in a cell (e.g., an immune cell such as an NK cell) which is at least 10-fold lower, at least 100-fold lower, at least 200-fold lower, at least 500-fold lower, or at least 1000-fold lower than the IL-18 polypeptide without the artificial polypeptide.

[0176] In some embodiments, the artificial polypeptide comprises a protease cleavage site. In some embodiments, the protease cleavage site is a site which is amenable to cleavage under certain specified or known conditions.

[0177] In some embodiments, the protease cleavage site is selected such that it is preferentially cleaved (e.g., cleaved at a faster rate or cleaved in more abundance) at a designated target tissue of a subject. In some embodiments, the protease cleavage site is preferentially cleaved at or near a target tissue of the subject such that the protease cleavage site is cleaved at a rate of least 2-fold, at least 4-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold greater than cleavage of the specific cleavage site at a different tissue. In some embodiments, the target tissue is diseased tissue of the subject. In some embodiments, the target tissue is cancer tissue of the subject. In some embodiments, the target tissue is a tumor microenvironment. In some embodiments, the target tissue is a tumor.

[0178] In some embodiments, the protease cleavage site is positioned such that all or only a portion of the artificial polypeptide is removed from the IL- 18 polypeptide after cleavage. In some embodiments, none of the artificial polypeptide is present on the IL- 18 polypeptide after cleavage (e.g., only residues corresponding to residues in SEQ ID NO: 701 are present after cleavage). In some embodiments, a portion of the artificial polypeptide remains attached to the IL-18 polypeptide after cleavage.

[0179] The artificial polypeptide can be attached to either the N-terminal residue or the C- terminal residue of the IL- 18 polypeptide. In some embodiments, the artificial polypeptide is attached to the N-terminal residue. In some embodiments, the artificial polypeptide is attached to the N-terminal amine of the IL-18 polypeptide. In some embodiments, the artificial polypeptide is attached to the C-terminal residue. In some embodiments, the artificial polypeptide is attachedWSGR Docket No. 56146-747.601 to the C-terminal carboxyl of the IL-18 polypeptide. In some embodiments, the N-terminal residue is the residue closest to residue position 1 of SEQ ID NO: 701 which is present on an IL-18 polypeptide as provided herein (e.g., the first residue of SEQ ID NO: 701 which has not been truncated). In some embodiments, the N-terminal residue of the IL-18 polypeptide is the residue at a position corresponding to position 1 in SEQ ID NO: 701. In some embodiments, the N- terminal residue of the IL- 18 polypeptide is Yl. In some embodiments, the N-terminal residue of the IL-18 polypeptide is Y1G. In some embodiments, the N-terminal residue of the IL-18 polypeptide is YIM. In some embodiments, the C-terminal residue is the residue at position 157 of SEQ ID NO: 701. In some embodiments, the C-terminal residue is D157.

[0180] In some embodiments, terminal residues of the IL- 18 polypeptide are substituted such that the artificial polypeptide is positioned such that the entirety of the artificial polypeptide is cleaved from the IL- 18 polypeptide. For example, if it is intended to introduce a cleavage site at a position corresponding to residue 1 of SEQ ID NO: 701, a protease cleavage sequence Pl-P2-P3-P’ l-P’2- P’3 can be selected (where the cleavage site is between P3 and P’ l), and residues 1, 2, and 3 of SEQ ID NO: 701 can be substituted for P’ l, P’2, and P’3 respectively, with P1-P2-P3- appended thereon. In this case, the artificial polypeptide would be considered to comprise P1-P2-P3-, with P’ 1, P’2, and P’3 as part of the IL-18 polypeptide (substituted residues).

[0181] In some embodiments, cleavage at the protease cleavage site leaves no amino acid residues attached to the IL- 18 polypeptide. In some embodiments, cleavage at the protease cleavage site leaves at least 1 amino acid residue attached to the IL- 18 polypeptide. In some embodiments, cleavage at the protease cleavage site leaves at most 1, 2, 3, 4, or 5 amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage at the protease cleavage site leaves 1, 2, 3, 4, or 5 amino acid residues attached to the IL-18 polypeptide. In some embodiments, cleavage at the protease cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues attached to the IL-18 polypeptide.

[0182] In some embodiments, the artificial polypeptide can be cleaved by a protease. In some embodiments, the artificial polypeptide contains a protease cleavage site that can be cleaved specifically by one or more proteases. In some embodiments, the protease cleavage site can be cleaved at a site preferred by one or more proteases.

[0183] In some embodiments, the protease is found at higher concentrations and / or demonstrates higher proteolytic activity at or near a target tissue of a subject. In some embodiments, the target tissue is disease tissue. In some embodiments, the target tissue is a cancer. In some embodiments, the target tissue is a tumor microenvironment.

[0184] In some embodiments, the protease is found at higher concentrations and / or demonstrates higher proteolytic activity at or near the tumor microenvironment relative to non-tumor tissue. InWSGR Docket No. 56146-747.601 some embodiments, the protease is found at higher concentrations at or near the tumor microenvironment relative to non-tumor tissue. In some embodiments, the protease demonstrates higher proteolytic activity at or near the tumor microenvironment relative to non-tumor tissue. In some embodiments, the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), granzyme M, a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease. In some embodiments, the cleavable peptide is cleavable by a protease selected from a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), granzyme M, a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a plasminogen activator, a cathepsin, a caspase, a tryptase, a matriptase, and a tumor cell surface protease, or any combination thereof. In some embodiments, the protease is selected from kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), granzyme M, urokinase plasminogen activator (uPA), a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a matriptase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease. In some embodiments, the cleavable peptide is cleavable by a protease selected from a kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), urokinase plasminogen activator (uPA), granzyme M, a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a matriptase, a plasminogen activator, a cathepsin, a caspase, a tryptase, a matriptase, and a tumor cell surface protease, or any combination thereof. In some embodiments, the protease is urokinase plasminogen activator (uPA) or matriptase. In some embodiments, the cleavable peptide is cleavable by urokinase plasminogen activator (uPA) or matriptase. In some embodiments, the protease is a protease selected from the Table 3 below.Table 3: Tissue / Tumor specific proteasesWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601

[0185] In some embodiments, the protease cleavage site is comprised within a protease recognition sequence. The protease recognition sequence can be recognized and cleaved by the protease. In some embodiments, the protease recognition sequence is a natural peptide sequence which has been incorporated into the artificial polypeptide. In some embodiments, the protease recognition sequence is a synthetic (e.g., man-made, designed, or engineered) sequence. In some embodiments, the protease recognition sequence comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to a sequence set forth in Table 3. Cleavage data for certain peptides of Table 4 can be found in WO2024150172A1.Table 4- Protease specific protease recognition sequencesWSGR Docket No. 56146-747.601

[0186] In some embodiments, cleavage of the protease cleavage site leaves no amino acid residues attached to the IL- 18 polypeptide. In such cases, the protease recognition sequence can be selected such that portions of the IL- 18 polypeptide make up part of the recognition sequence, or are compatible therewith. For example, in some embodiments, the sequence PLG is appended to the N-terminus of the IL-18 polypeptide (e.g., residue 1 of SEQ ID NO: 701), which results in the specific cleavage site being between the G of the PLG and the N-terminus of the IL-18 polypeptide, thereby resulting in a “scarless” activated IL- 18 polypeptide after cleavage.

[0187] In some embodiments, a portion of the protease recognition sequence which defines the protease cleavage site will be comprised in the sequence of the IL-18 polypeptide (e.g., part of the protease recognition sequence will be comprised at positions which correspond to positions of SEQ ID NO: 701). In some embodiments, the portion of the protease recognition sequence comprised in the sequence of the IL- 18 polypeptide will be substituted relative to the sequence set forth in SEQ ID NO: 701. For example, in some embodiments, the last three amino acids of SEQ ID NO: 701 are substituted with -PLG in order to form part of a protease recognition site with the artificial polypeptide. By way of further non-limiting examples, SEQ ID NOs: 747, 748, 749, 750, and 754 form a protease recognition site with the N-terminal YFG of an IL-18 polypeptide as described herein such that, if that particular cleavage site is cleaved, the entirety of the artificial polypeptide is cleaved from the IL-18 polypeptide.

[0188] In some embodiments, cleavage of the protease cleavage site leaves at least 1 amino acid residue attached to the IL-18 polypeptide. In some embodiments, cleavage of the protease cleavage site leaves at most 1, 2, 3, 4, or 5 amino acid residues attached to the IL- 18 polypeptide. In some embodiments, cleavage of the protease cleavage site leaves 1, 2, 3, 4, or 5 amino acid residues attached to the IL- 18 polypeptide. In some embodiments, cleavage of the protease cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acid residues attached to the IL- 18 polypeptide. In some embodiments, cleavage of the protease cleavage site leaves at most 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues attached to the IL-18 polypeptide.Further exemplary protease recognition sequence, such as cleavable peptide sequences which can be incorporated into an Act-IL-18 polypeptide as provided herein can be found in any one of U.S. Patent Publication Nos: US2010 / 0189651, US2016 / 0289324, US2018 / 0125988,US2019 / 0153115, US2020 / 0385469, US2021 / 0260163, US2022 / 0048949, US2022 / 0267400,WSGR Docket No. 56146-747.601US2021 / 0115102, US2022 / 0002370, US2021 / 0163562, US20200392235, US2021 / 0139553, US2021 / 0317177, US2020 / 0283489, US2021 / 0002343, US2021 / 0292421, US2021 / 0284728, US2021 / 0269530, US2022 / 0054544, US2021 / 0355219, US2022 / 0073613, US2021 / 0047406, and / or Patent Cooperation Treaty Publication Nos: WO2021 / 202675, W02021 / 062406, WO2021 / 142471, WO2021 / 216468, WO2021 / 119516, WO2021 / 253360, WO2021 / 146455, WO202 1 / 202678, WO2021 / 202673, WO2021 / 189139, W02020 / 232303, WO2022 / 115865,WO202 1 / 202678, and / or Chen et. al., J Bio Chem, 277, V6 P4485-4491 (2002). Certain preferred cleavable peptide sequences and cleavage data for these cleavable peptide sequence can be found in WO2024150172A1, the contents of which are incorporated herein by reference.

[0189] An artificial polypeptide can comprise a group such as polymer or spacer (e.g., a spacer peptide) used to link the artificial moiety to the IL- 18 polypeptide.Blocking Moieties

[0190] In some embodiments, the artificial polypeptide comprises a blocking moiety. In some embodiments, the blocking moiety is a group which, when attached to the IL-18 polypeptide in the Act-IL-18 polypeptide, acts to disrupt or inhibit binding of the IL-18 polypeptide with the IL- 18 receptor or a subunit thereof (e.g., as measured by experiments designed to detect binding, or by in vitro or in vivo activity analysis of the Act-IL-18 polypeptide).

[0191] In some embodiments, the blocking moiety is a steric blocking group or a specific blocking group. In some embodiments, the blocking moiety is a steric blocking group. In some embodiments, a steric blocking group has no specific interaction with the IL- 18 polypeptide, but its presence hinders the interaction of the Act-IL-18 polypeptide with the receptor owing to its bulk. In some embodiments, the steric blocking group is a polymer (e.g., polyethylene glycol) or a polypeptide (e.g., albumin, an Fc region, etc.).

[0192] In some embodiments, the blocking moiety is a specific blocking group. In some embodiments, the specific blocking group has a specific binding or other interaction to IL- 18. Non-limiting examples of specific blocking groups can include IL- 18 propeptides, antibodies or antigen binding fragments which bind IL- 18, IL- 18 receptor subunits or domains or other fragments thereof, IL- 18 binding proteins or fragments thereof, or other groups capable of specific binding to IL- 18.

[0193] In some embodiments, the blocking moiety is a propeptide of IL-18, or a variant thereof. In some embodiments, the blocking moiety is a propeptide of IL-18. Endogenously, human IL-18 is expressed as an immature, inactive 193 amino acid having the sequence MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNKDTKSDIIFFQRS VPGHDNKMQFES S S YEGYFL ACEKERDLFKLILKKEDELGWSGR Docket No. 56146-747.601DRSIMFTVQNED (SEQ ID NO: 700), the first 36 amino acids of which are a propeptide which is cleaved by caspases to yield the mature, active form of IL-18 (SEQ ID NO: 701). In some embodiments, the blocking moiety is attached to the N-terminus of the IL-18 polypeptide (e.g., through a cleavable peptide comprising the specific cleavage site and any optional linker peptides), and the blocking moiety is a propeptide of IL-18. In some embodiments, the blocking moiety is a human IL-18 propeptide or a variant thereof. In some embodiments, the blocking moiety is an IL-18 propeptide an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 731. In some embodiments, the blocking moiety is an IL- 18 propeptide having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 731. In some embodiments, the blocking moiety is an IL-18 propeptide comprising the sequence set forth in SEQ ID NO: 731. In some embodiments, the blocking moiety is an IL-18 propeptide comprising the sequence set forth in SEQ ID NO: 731 with a substitution for residue CIO (e.g., a CIOS substitution (SEQ ID NO: 732) or a C10A substitution (SEQ ID NO: 733). In some embodiments, the IL-18 propeptide comprises one or more modifications (e.g., amino acid substitutions) which reduce the affinity of the IL-18 propeptide for the IL-18 polypeptide in the Act-IL-18 polypeptide. In some embodiments, the blocking moiety is an IL-18 propeptide, and the protease cleavage site of the Act-IL-18 polypeptide is different from that of the endogenous propeptide (i.e., the specific cleavage site is not the bond between D36 and Y37 of SEQ ID NO: 700). In some embodiments, the IL-18 propeptide acting as a blocking moiety is connected to the N-terminus of the IL- 18 polypeptide in the Act-IL-18 polypeptide through another protease recognition sequence (and optionally one or more linking peptides).

[0194] In some embodiments, the blocking moiety is a modified IL- 18 propeptide (e.g., human IL- 18 propeptide) directly attached to the N-terminus of the IL- 18 polypeptide. In some embodiments, the modified IL-18 propeptide comprises modifications which change the natural caspase cleavage site of SEQ ID NO: 700 to a site cleaved by another protease (e.g., any of the proteases provided herein, such as a matrix metalloprotease). In some embodiments, the three C- terminal amino acids of the IL-18 propeptide are substituted to -PLG (e.g., SEQ ID NOs: 734, 735, and 736). In some embodiments, the IL-18 propeptide comprises the -PLG substitution and at least one of the first 3 residues of the IL-18 polypeptide is substituted relative to SEQ ID NO: 501 to make a complete protease recognition sequences. In some embodiments, the first 3 residues of the IL-18 polypeptide are YFG (i.e., not substituted relative to wild type IL-18) and yet a protease recognition site is still formed with the terminus of the IL- 18 propeptide.

[0195] In some embodiments, the IL-18 propeptide blocking moiety is a truncated version of an IL- 18 propeptide described herein. In some embodiments, the IL- 18 propeptide blocking moietyWSGR Docket No. 56146-747.601 comprises a truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 residues off of the N-terminus relative to SEQ ID NO: 731. In some embodiments, the IL-18 propeptide comprises a truncation of up to 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 amino acids from the N-terminus relative to SEQ ID NO: 731. In some embodiments, the IL- 18 propeptide blocking moiety comprises a truncation of 8 residues from the N-terminus relative to SEQ ID NO: 731 (e.g., as in SEQ ID NO: 737). In some embodiments, the IL-18 propeptide blocking moiety comprises a truncation of 13 residues from the N-terminus relative to SEQ ID NO: 731 (e.g., as in SEQ ID NO: 738). In some embodiments, the IL-18 propeptide blocking moiety comprises a truncation of 26 residues from the N-terminus relative to SEQ ID NO: 731 (e.g., as in SEQ ID NO: 739). In some embodiments, the IL- 18 propeptide blocking moiety has a sequence at least 80%, 85%, 90%, 95%, or 100% identical to the sequence set forth in any one of SEQ ID NOs: 737, 738, or 739.

[0196] In some embodiments, the IL-18 propeptide comprises particular amino acid substitutions relative to SEQ ID NO: 731. In some embodiments, the IL-18 propeptide comprises substitutions at one or more of the following residues relative to SEQ ID NO: 731 : CIO, M16, F18, L23, and / or F25. In some embodiments, the IL-18 propeptide comprises 1, 2, 3, 4, or 5 substitutions at residues CIO, M16, F18, L23, and / or F25. In some embodiments, the IL-18 propeptide comprises substitutions at one or more of the following residues relative to SEQ ID NO: 731 : M16, F18, L23, and / or F25. In some embodiments, the IL-18 propeptide comprises 1, 2, 3, or 4 substitutions at residues M16, F18, L23, and / or F25. In some embodiments, the IL-18 propeptide comprises a substitution at residue CIO. In some embodiments, the IL- 18 propeptide comprises a C10A or CIOS substitution. In some embodiments, the IL- 18 propeptide comprises a substitution at residue M16. In some embodiments, the M16 substitution is M16A. In some embodiments, the IL-18 propeptide comprises a substitution at residue Fl 8. In some embodiments, the F18 substitution is F18A. In some embodiments, the IL-18 propeptide comprises a substitution at residue L23. In some embodiments, the L23 substitution is L23A. In some embodiments, the IL- 18 propeptide comprises a substitution at residue F25. In some embodiments, the F25 substitution is F25. In some embodiments, the IL-18 propeptide comprises M16A, F18A, L23A, and F25A substitutions. In some embodiments, the IL-18 propeptide comprises C10A, M16A, F18A, L23A, and F25A substitutions. In some embodiments, the IL-18 propeptide comprises CIOS, M16A, F18A, L23A, and F25A substitutions. In some embodiments, the substitutions listed in this paragraph are incorporated into a truncated IL-18 propeptide described above (e.g., into any one of SEQ ID NOs: 737, 738, or 739). In some embodiments, the IL-18 propeptide comprises the sequence set forth in SEQ ID NO: 740.Table 5 - Exemplary IL-18 Propeptide SequencesWSGR Docket No. 56146-747.601

[0197] In some embodiments, an Act-IL-18 polypeptide comprising a human IL- 18 propeptide or variant thereof as a blocking moiety exhibits substantially reduced activity compared to the IL- 18 polypeptide by itself or the activated form of the IL- 18 polypeptide (e.g., substantially no activity, or activity which is reduced by more than 1000-fold). In some embodiments, it may be desirable for the Act-IL-18 with an IL- 18 propeptide attached to retain a greater activity prior to activation. In order to accomplish this, in some embodiments, it may be advantageous to use an IL-18 propeptide from a non-human species. In some embodiments, an Act-IL-18 polypeptide provided herein comprises an IL- 18 propeptide from a non-human species. In some embodiments, the non-human species is a mammal. In some embodiments, the non-human species is a primate, a rodent, an equine, a bovine, an urcine, a porcine, an equine, a chiroptera, a camelid, or other animal. In some embodiments, the non-human IL-18 propeptide has an amino acid sequence which is at least 50%, 60%, 70%, or 75% identical to that of SEQ ID NO: 731.

[0198] In some embodiments, the blocking moiety comprises a portion (e.g., a domain or portion thereof) of an IL- 18 receptor subunit, or a variant thereof. In some embodiments, the portion of the IL- 18 receptor subunit or variant thereof is attached to the C-terminus of the IL- 18 polypeptide in the Act-IL-18 polypeptide (e.g., through a cleavable peptide comprising the specific cleavage site and any optional linker peptides). In some embodiments, the blocking moiety comprises a portion of the IL- 18 receptor alpha subunit or the IL- 18 receptor beta subunit, or a variant thereof. In some embodiments, the blocking moiety comprises a portion of the IL-18 receptor alpha subunit or a variant thereof. In some embodiments, the blocking moiety comprises a domain of the IL-18 receptor alpha subunit or a variant thereof. In some embodiments, the blocking moiety comprises an extracellular domain, or a variant thereof, of the IL- 18 receptor alpha subunit. InWSGR Docket No. 56146-747.601 some embodiments, the blocking moiety comprises the DI, D2, orD3 domain, or a variant thereof, of the IL- 18 receptor alpha subunit.

[0199] In some embodiments, the blocking moiety comprises the D3 domain, or a variant thereof, of the IL- 18 receptor alpha subunit. The sequence of the human D3 domain of the IL- 18 receptor alpha subunit is shown in SEQ ID NO: 715 below. In some embodiments, the blocking moiety comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 715. In some embodiments, the blocking moiety comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 715. In some embodiments, the blocking moiety comprises the sequence set forth in SEQ ID NO: 715. In some embodiments, the blocking moiety D3 domain of the IL- 18 receptor alpha subunit comprises substitutions which remove glycosylation sites from the D3 domain. In some embodiments, the blocking moiety comprises the amino acid sequence set forth in SEQ ID NO: 716 below.Table 6 - Additional IL-18 Blocking Moieties

[0200] In some embodiments, it may be desirable to introduce one or more substitutions of amino acids to the sequence of the D3 domain of the IL- 18 receptor alpha subunit when used as a blocking moiety in order to enhance or detune the binding of the D3 domain to the IL- 18 polypeptide in the Act-IL-18 polypeptide.

[0201] The full length human IL- 18 receptor alpha subunit has the sequence MNCRELPLTLWVLISVSTAESCTSRPHITVVEGEPFYLKHCSCSLAHEIETTTKSWYKSS GSQEHVELNPRSSSRIALHDCVLEFWPVELNDTGSYFFQMKNYTQKWKLNVIRRNKHS CFTERQVTSKIVEVKKFFQITCENSYYQTLVNSTSLYKNCKKLLLENNKNPTIKKNAEFE DQGYYSCVHFLHHNGKLFNITKTFNITIVEDRSNIVPVLLGPKLNHVAVELGKNVRLNC SALLNEEDVIYWMFGEENGSDPNIHEEKEMRIMTPEGKWHASKVLRIENIGESNLNVLY NCTVASTGGTDTKSFILVRKADMADIPGHVFTRGMIIAVLILVAVVCLVTVCVIYRVDL VLFYRHLTRRDETLTDGKTYDAFVSYLKECRPENGEEHTFAVEILPRVLEKHFGYKLCIF ERDVVPGGAVVDEIHSLIEI<SRRLIIVLSI<SYMSNEVRYELESGLHEALVERI<II<IILIEFTWSGR Docket No. 56146-747.601PVTDFTFLPQSLKLLKSHRVLKWKADKSLSYNSRFWKNLLYLMPAKTVKPGRDEPEVL PVLSES (SEQ ID NO: 717) (UniProt ID: Q13478).Linking Peptides

[0202] In some embodiments, the artificial polypeptide comprises one or more linking peptides. In some embodiments, the linking peptide of an artificial polypeptide is positioned between the protease cleavage site and the IL-18 polypeptide (i.e., the linking peptide remains attached to the IL-18 polypeptide after cleavage) or is positions between the protease cleavage site and a blocking moiety, or both (e.g., the Act-IL-18 polypeptide has two linking peptides). In some embodiments, a linking peptide comprises from 1 to 50 amino acid, from 1 to 40 amino acids, from 1 to 30 amino acids, from 1 to 25 amino acids, from 1 to 20 amino acids, from 1 to 15 amino acids, from 1 to 10 amino acids, or from 1 to 5 amino acids. In some embodiments, the linking peptide comprises from 1 to 15 amino acids. In some embodiments, the linking peptide consists of amino acids glycine and serine. In some embodiments, the linking peptide consists of glycines. Non-limiting examples of a linking peptide include, but are not limited to (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), or (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, a linking peptide can be GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29) or (GGGGS)4 (SEQ ID NO: 30). Additional linking peptides can include GGGGS GGGGS GGGG (SEQ ID NO: 712). Linking peptides can also be positioned between the base sequence of an IL- 18 polypeptide and a sortase tag as described herein. In embodiments where the artificial polypeptide comprises multiple linking peptides, each linking peptide can be the same or different.Act IL- 18 Molecular Orientations

[0203] Act-IL-18 polypeptides provided herein can have a variety of orientations. In some embodiments, an Act-IL-18 polypeptide provided herein comprises an orientation according to any one of the below formulas:(a) IL-18-CS;(b) IL-18-LPi-CS;(c) IL-18 - LP1-CS-LP2-BM;(d) IL-I8-CS-LP1-BM;(e) IL-18-CS-BM;(f) IL-I8-LP1-CP-BM;(g) BM-CS-IL-18;(h) BM-LP1-CS-IL-I8;(i) BM-LP1-CS-LP2-IL-I8;WSGR Docket No. 56146-747.601(j) BM-CS-LPi-IL-18;(k) CS-LPi-IL-18; and(l) CS-IL-18; wherein IL-18 is the IL-18 polypeptide, CS is a protease recognition sequence (e.g., any of the protease recognition sites described herein), LPi is a first linking peptide, LP2 is a second linking peptide, and BM is a blocking moiety, and wherein the orientations are shown in an N-terminal to C-terminal order.

[0204] In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (a).In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (b). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (c). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (d). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (e). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (f). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (g). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (h). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (i). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (j). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (k). In some embodiments, the Act-IL-18 polypeptide comprises the orientation of formula (1).

[0205] In the orientations described above, it is expressly contemplated herein that the IL-18 polypeptide can be any of the IL-18 polypeptides described herein, the CS moiety can be any of the cleavage sequences described herein (e.g., any of those in Table 3, or one having a specific sequence identity thereto, or another cleavage sequence as described herein), the blocking moi eties can be any of the blocking moi eties described herein (e.g., any of the IL- 18 propeptides as described herein), and the linking peptides can be any of the linking peptides described herein, independently of any of the other components.

[0206] In certain preferred embodiments, the Act-IL-18 polypeptides as described herein are of the formula (g) BM-CS-IL-18. In some embodiments, the blocking moiety is an IL- 18 propeptide as described herein (e.g., any of the IL-18 propeptides described herein, including any of the variants described herein). In some embodiments, the CS group is one of the cleavage sequence described herein (e.g., any of those in Table 3, or any variant thereof described herein). In some embodiments, the IL-18 polypeptide is any one of those described herein (e.g., any one of those set forth in the Tables below which described IL-18 polypeptide sequences, or another IL-18 variant polypeptide described herein).Active Form of the Act-IL-18 polypeptideWSGR Docket No. 56146-747.601

[0207] In some embodiments, cleavage of the protease cleavage site of the artificial polypeptide converts the Act-IL-18 into an active form of the IL- 18 polypeptide. As used herein, the active form of the IL- 18 polypeptide refers to the cleaved version of the IL- 18 polypeptide which possesses some or all of the activity associated with the IL- 18 polypeptide which is inactivated by the artificial polypeptide. Additionally, unless context clearly indicates otherwise, reference to simply “the IL- 18 polypeptide” refers to an IL- 18 polypeptide which was never prepared in an activatable form (E.g., it refers to the base IL- 18 polypeptide on which an Act-IL-18 polypeptide is based(i.e., without the artificial polypeptide attached). In some embodiments, the active form of the IL- 18 polypeptide comprises a portion the artificial polypeptide still attached to the IL- 18 polypeptide (e.g., a subset of amino acid residues of the artificial polypeptide). In some embodiments, the active form of the IL-18 polypeptide is the same as the IL-18 polypeptide (e.g., has the same amino acid sequence as the IL- 18 polypeptide because the entire artificial polypeptide has been cleaved).

[0208] In some embodiments, the active form of the IL- 18 polypeptide provided herein displays reduced binding to the IL- 18 binding protein (IL-18BP) relative to WT-IL-18. The active form of the IL- 18 polypeptides may also display binding characteristics for the IL-18Rab that differ from wild-type IL- 18 (e.g., a higher affinity for the IL-18Rab heterodimer or a lower affinity for the IL-18Rab heterodimer). In preferred embodiments, the affinity for IL-18Rab of the active form of the IL-18 polypeptide is not substantially lower than the affinity of WT IL-18 for IL-18Rab (e.g., the active form of the Act-IL-18 polypeptide’s affinity for IL-18Rab is no less than about 500x lower than wild type IL- 18).

[0209] In some embodiments, the active form of the IL-18 polypeptides displays increased affinity for an IL- 18 receptor alpha subunit (IL-18Ra) or an IL- 18 receptor beta subunit (IL-18Rb) relative to wild type IL-18. In some embodiments, the active form of the IL-18 polypeptides have an increased affinity for the IL-18Ra / b heterodimer relative to IL- 18 WT. In one aspect, the active form of the IL- 18 polypeptides described herein have decreased affinity for the IL-18Ra / b heterodimer relative to wild type IL- 18.

[0210] In some embodiments, the binding affinity between the active form of the IL- 18 polypeptides and IL-18Ra is the same as or lower than the binding affinity between a wild-type IL-18 and IL-18Ra. In some embodiments, the binding affinity between the active form of the IL- 18 polypeptides and IL-18Ra is the same as or higher than the binding affinity between a wildtype IL-18 and IL-18Ra. In some embodiments, the binding affinity between the active form of the IL- 18 polypeptides and IL-18Rb is the same as or lower than the binding affinity between a wild-type IL- 18 and IL-18Rb. In some embodiments, the binding affinity between the active form of the IL- 18 polypeptides and IL-18Rb is the same as or higher than the binding affinity betweenWSGR Docket No. 56146-747.601 a wild-type IL- 18 and IL-18Rb. In some embodiments, the binding affinity between the active form of the IL- 18 polypeptides and the IL-18Ra / b heterodimer is the same as or lower than the binding affinity between a wild-type IL- 18 and the IL-18Ra / b heterodimer. In some embodiments, the binding affinity between the active form of the IL-18 polypeptides and the IL-18Ra / b heterodimer is the same as or higher than the binding affinity between a wild-type IL-18 and the IL-18Ra / b heterodimer.

[0211] In some embodiments, an active form of the IL- 18 polypeptide provided herein displays an ability to induce interferon gamma (IFNy) production after administration to a subject. In some embodiments, the ability to induce IFNY is comparable to that of a wild type IL-18 (e.g., displays an EC50 for fFNy induction that is within about 10-fold of that of a wild type IL-18).

[0212] In some embodiments, an active form of the Act-IL-18 polypeptide provided herein also display a reduced binding IL-18 binding protein (IL-18BP). In some embodiments, the active form of the IL- 18 polypeptide provided herein can induce IFNy even in the presence of IL-18BP (e.g., the ability of the active form of the Act-IL-18 polypeptide to induce IFNy is not substantially inhibited by the presence of IL-18BP).

[0213] In some embodiments, an active form of IL-18 polypeptide provided herein displays a significant reduction in inhibition of the ability to induce IFNy production in the presence of IL- 18BP compared to wild type IL-18. In some embodiments, an active form of IL-18 polypeptide provided herein displays a similar or only slightly reduced ability to induce IFNy production compared to wild type IL-18, and a significant reduction in inhibition of the ability to induce IFNy production in the presence of IL-18BP compared to wild type IL-18.Biological activity of Act-IL- 18 and the Active Form of the IL-18 Polypeptide

[0214] In some embodiments the Act-IL-18 exhibits a decreased affinity for the IL-18 receptor of at least 10-fold, at least 100 fold, at least 500 fold, at least 1000 fold lower in comparison to wild type IL- 18 or the active form of the IL- 18 polypeptide. In some embodiments, the Act-IL- 18 exhibits an increased EC50 for the production of fFNy that is at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold higher in comparison to wild type IL- 18 or the active from of the IL-18 polypeptide. In some embodiments, the Act-IL-18 exhibits an increased EC50 for the production of IFNy that is at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000 fold higher in comparison to the active form of the IL-18 polypeptide. In some embodiments, the Act-IL-18 exhibits an increased EC50 for the production of IFN-g that is at least 5 fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold higher in comparison to the IL- 18 polypeptide.

[0215] In some embodiments, the active form of the IL- 18 polypeptide provided herein exhibits reduced affinity for IL-18 binding protein (IL-18BP) compared to WT IL-18 (SEQ ID NO: 701).WSGR Docket No. 56146-747.601In some embodiments, the active form of the IL-18 polypeptide exhibits at least 2-fold, at least 5- fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60- fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold lower affinity for IL- 18BP compared to WT IL- 18. In some embodiments, the active form of the IL- 18 polypeptide exhibits at least a 10-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the active form of the IL-18 polypeptide exhibits at least a 20-fold lower affinity for IL-18BP compared to WT IL- 18. In some embodiments, the active form of the IL- 18 polypeptide exhibits at least a 50-fold lower affinity for IL-18BP compared to WT IL-18. In some embodiments, the active form of the IL- 18 polypeptide exhibits at least an 80-fold lower affinity for IL-18BP compared to WT IL- 18. In some embodiments, the active form of the IL- 18 polypeptide exhibits at least a 100-fold lower affinity for IL-18BP compared to WT IL-18.

[0216] In some embodiments, the active form of the IL- 18 polypeptide provided herein exhibits a reduced binding to IL-18BP as measured by KD. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18BP of at least about 1 nM, at least about 5 nM, at least about 10 nM, at least about 15 nM, at least about 20 nM, at least about 25 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, or at least about 500 nM. In some embodiments, the active form of the IL-18 polypeptide exhibits a KD with IL-18BP of at least about 1 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18BP of at least about 5 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18BP of at least about 50 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18BP of at least about 100 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18BP of at least about 500 nM.

[0217] In some embodiments, the active form of the IL- 18 polypeptide displays at most an only slightly diminished affinity for IL-18Rab compared to WT IL-18 (SEQ ID NO: 701). In some embodiments, the active form of the IL-18 polypeptide exhibits at most a 2-fold lower, at most a 3-fold lower, at most a 4-fold lower, at most 5-fold lower, at most a 10-fold lower, at most a 15- fold lower, at most a 20-fold lower, at most a 30-fold lower, at most a 40-fold lower, at most a 50- fold lower, at most a 75 -fold lower, or at most a 100-fold lower affinity for IL-18 Rab as compared to the affinity of WT IL-18 for IL-18Rab. In some embodiments, the active form of the IL-18 polypeptide exhibits at most a 10-fold lower affinity for IL-18Rab as compared to the affinity of WT IL- 18 for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 20-fold lower affinity for IL-18Rab as compared to the affinity of WT IL-18 for IL- 18Rab. In some embodiments, the active form of the IL-18 polypeptide exhibits at most a 50-fold lower affinity for IL-18Rab as compared to the affinity of WT IL- 18 for IL-18Rab. In someWSGR Docket No. 56146-747.601 embodiments, the active form of the IL- 18 polypeptide exhibits at most a 100-fold lower affinity for IL- 18 Rab as compared to the affinity of WT IL- 18 for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits an increased affinity for IL-18Rab compared to WT IL-18. In some embodiments, the affinity is increased by at least 2-fold, at least 4-fold, at least 6- fold, at least 8-fold, or at least 10-fold compared to WT IL-18.

[0218] In some embodiments, the active form of the IL- 18 polypeptide displays at most an only slightly diminished affinity for IL-18Rab compared to the corresponding IL- 18 polypeptide without the artificial polypeptide. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 2-fold lower, at most a 3-fold lower, at most a 4-fold lower, at most 5-fold lower, at most a 10-fold lower, at most a 15-fold lower, at most a 20-fold lower, at most a 30-fold lower, at most a 40-fold lower, at most a 50-fold lower, at most a 75-fold lower, or at most a 100- fold lower affinity for IL-18 Rab as compared to the affinity of the corresponding IL-18 polypeptide without the artificial polypeptide for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 2-fold lower affinity for IL-18Rab as compared to the affinity of the corresponding IL- 18 polypeptide without the artificial polypeptide for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 3 -fold lower affinity for IL-18Rab as compared to the affinity of the corresponding IL- 18 polypeptide without the artificial polypeptide for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 4-fold lower affinity for IL-18Rab as compared to the affinity of the corresponding wild type IL- 18 polypeptide without the N-terminal domain for IL-18Rab. In some embodiments, the active form of the IL-18 polypeptide exhibits at most a 5-fold lower affinity for IL- 18 Rab as compared to the affinity of the corresponding IL- 18 polypeptide without the artificial polypeptide for IL-18Rab. In some embodiments, the active form of the IL- 18 polypeptide exhibits at most a 10-fold lower affinity for IL-18Rab as compared to the affinity of the corresponding IL- 18 polypeptide without the artificial polypeptide for IL-18Rab.

[0219] In some embodiments, the active form of the IL- 18 polypeptide provided herein exhibits at most only a slight reduction in binding to IL-18Rab as measured by KD. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18Rab of at most about 10 nM, at most about 20 nM, at most about 30 nM, at most about 50 nM, at most about 75 nM, at most about 100 nM, or at most about 200 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18Rab of at most about 20 nM. In some embodiments, the active form of the Act-IL-18 polypeptide exhibits a KD with IL-18Rab of at most about 30 nM. In some embodiments, the active form of the IL- 18 polypeptide exhibits a KD with IL-18Rab of at most about 40 nM. In some embodiments, the active form of the IL-18 polypeptide exhibits a KD with IL-18Rab of at most about 50 nM. In some embodiments, the active form of the IL-18 polypeptideWSGR Docket No. 56146-747.601 exhibits an increase in binding to IL-18Rab compared to WT IL-18 as measured by KD. In some embodiments, the active form of the IL- 18 polypeptide has a KD with IL-18Ra of at most about 2 nM, at most about 1 nM, at most about 0.5 nM, or at most about 0.2 nM.

[0220] In some embodiments, the active form of the IL- 18 polypeptide exhibits a wide window in which the active form of the IL- 18 polypeptide will bind to IL-18Rab even in the presence of IL-18BP. In some embodiments, this window can be measured by a ratio of KD of the Act-IL- 18 / IL-18BP interaction over KD of the IL-18 / IL-18Rab interaction, where a larger number indicates a larger window in which the active form of the Act-IL-18 polypeptide is expected to be active in vivo. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL-18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio ofKD of the IL-18 / IL-18BP interaction overKD of the IL-18 / IL-18Rab interaction of at least about 2. In some embodiments, the active form of the IL-18 polypeptide exhibits a ratio of KD of the IL-18 / IL-18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 5. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL-18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 10. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL- 18 / IL-18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 25. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL- 18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 30. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL- 18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 40. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL- 18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 45. In some embodiments, the active form of the IL- 18 polypeptide exhibits a ratio of KD of the IL-18 / IL- 18BP interaction over KD of the IL-18 / IL-18Rab interaction of at least about 50.

[0221] In some embodiments, the active form of the IL- 18 polypeptides provided herein display one or more activities associated with WT IL-18. In some embodiments, the active form of the IL- 18 polypeptide exhibits a similar ability to signal through the IL- 18 receptor (IL-18Rab) but lacks the ability or displays a reduced ability to be inhibited by IL-18BP. In some embodiments, the active form of the IL-18 polypeptide’s ability to signal through IL-18Rab is reduced compared to WT IL- 18 by only a small amount.WSGR Docket No. 56146-747.601

[0222] In some embodiments, the active form of the IL-18 polypeptide modulates IFNy production when in contact with a cell (e.g., an immune cell, such as an NK cell). In some embodiments, the active form of the IL- 18 polypeptide’s ability to modulate IFNY production is measured as a half-maximal effective concentration (EC50). In some embodiments, an EC50 (nM) of the active form of the Act-IL-18 polypeptide’s ability to induce IFNy is less than 10-fold higher than, less than 5-fold higher than, or less than an EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 of the active form of the Act-IL-18 polypeptide’s ability to induce IFNy is less than 10-fold higher than an EC50 (nM) of an IL- 18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 of the active form of the Act-IL-18 polypeptide’s ability to induce IFNY is less than 5-fold higher than an EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy is less than an EC50 (nM) of an IL- 18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 of the active form of the IL- 18 polypeptide’s ability to induce IFNy is less than 10-fold higher than, less than 8-fold higher than, less than 6-fold higher than, less than 5-fold higher than, less than 4-fold higher than, less than 3-fold higher than, or less than 2-fold higher than an EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy is measured by an IFNy induction cellular assay.

[0223] In some embodiments, a half-maximal effective concentration (ECso) of the ability of the active form of the IL- 18 polypeptide to stimulate production of IFNy is at most 100-fold higher than, at most 50-fold higher than, at most 20-fold higher than, at most 10-fold higher than, at most 5-fold higher than, or at most 2-fold higher than that of a WT IL-18 (SEQ ID NO: 701). In some embodiments, the ability of the active form of the IL- 18 polypeptide to stimulate IFNy production is enhanced compared to WT IL-18. In some embodiments, a half-maximal effective concentration (ECso) of the ability of the active form of the IL-18 polypeptide to stimulate production of IFNy is at least 5-fold lower than, at least 10-fold lower than, at least 20-fold lower than, at least 50-fold lower than, at least 75-fold lower than, or at least 100-fold higher than that of a WT IL-18.

[0224] In some embodiments, an EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy production is less than about 100 nM, less than about 75 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 15 nM, or less than about 10 nM. In some embodiments, an EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy production is less than about 100 nM. In some embodiments, an EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy production is lessWSGR Docket No. 56146-747.601 than about 50 nM. In some embodiments, an EC50 of the active form of the IL-18 polypeptide’s ability to induce IFNy production is less than about 10 nM.

[0225] In some embodiments, the active form of the IL- 18 polypeptide exhibits a reduced ability to have its IFNY induction activity inhibited by IL-18BP compared to WT IL- 18. In some embodiments, the active form of the IL-18 displays a half-maximal inhibitory concentration (IC50) by IL-18BP which is at least about 10-fold higher than, at least about 20-fold higher than, at least about 50-fold higher than, at least about 75-fold higher than, at least about 100-fold higher than, at least about 200-fold higher than, at least about 300-fold higher than, at least about 400- fold higher than, at least about 500-fold higher than, at least about 600-fold higher than, at least about 700-fold higher than, at least about 800-fold higher than, at least about 900-fold higher than, or at least about 1000-fold higher than an IC50 of WT IL-18’s inhibition by IL-18BP. In some embodiments, the active form of the IL-18 displays a half-maximal inhibitory concentration (IC50) by IL-18BP which is at least about 100-fold higher than an IC50 of WT IL-18’s inhibition by IL-18BP. In some embodiments, the active form of the IL-18 displays a half-maximal inhibitory concentration (IC50) by IL-18BP which is at least about 500-fold higher than an IC50 of WT IL-18’s inhibition by IL-18BP. In some embodiments, the active form of the IL-18 displays a half-maximal inhibitory concentration (IC50) by IL-18BP which is at least about 1000-fold higher than an IC50 of WT IL-18’ s inhibition by IL-18BP.

[0226] In some embodiments, the active form of the IL- 18 polypeptide exhibits a favorable ratio of half-maximal inhibitory concentration (IC50) by IL-18BP over a half-maximal effective concentration (EC50) of IFNy induction (IC50ZEC50 ratio). In some embodiments, the IC50ZEC50 ratio is increased compared to WT IL-18. In some embodiments, the IC50ZEC50 ratio is increased by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, or at least about 1000-fold compared to WT IL-18. In some embodiments, the IC50ZEC50 ratio is increased by at least about 10-fold compared to WT IL-18. In some embodiments, the IC50 / EC50 ratio is increased by at least about 100-fold compared to WT IL-18. In some embodiments, the IC50ZEC50 ratio is increased by at least about 500-fold compared to WT IL-18. In some embodiments, the IC50ZEC50 ratio of the active form of the Act-IL-18 polypeptide is at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 250, or at least about 500.

[0227] In some embodiments, the active form of the IL-18 polypeptide modulates IFNy production, and wherein an EC50 (nM) of the active form of the Act-IL-18 polypeptide against IFNy is less than an EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In someWSGR Docket No. 56146-747.601 embodiments, the EC50 (nM) of the active form of the Act-IL-18 polypeptide against IFNy is at least 10-fold less than the EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 (nM) of the active form of the Act-IL-18 polypeptide against IFNy is about 10-fold less than the EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701. In some embodiments, the EC50 (nM) of the active form of the Act-IL-18 polypeptide against IFNY is about 15-fold less than the EC50 (nM) of an IL-18 polypeptide of SEQ ID NO: 701.

[0228] In some embodiments, the activated form of the IL-18 polypeptide (e.g., after cleavage of the specific cleavage site) exhibits an enhanced activity associated with IL-18 compared to the Act-IL-18 polypeptide with the specific cleavage site intact. In some embodiments, the active form of the IL-18 polypeptide exhibits an activity which is enhanced by at least 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold, 10000-fold, 15000-fold, or 20,000-fold higher than the Act-IL-18 polypeptide. Such activities can include induction of production of IFNy in a cell (e.g., an immune cell such as an NK cell), activation of signaling through the IL- 18 receptor (e.g., in a reporter assay), or another in vitro or in vivo activity. In some embodiments, the activated form of the IL- 18 polypeptide exhibits enhanced binding to the IL- 18 receptor or a subunit thereof (e.g., the IL- 18 receptor alpha subunit) compared to the Act-IL-18 polypeptide (e.g., has a KD which is at least 10-fold, 20-fold, 50-fold, or 100- fold lower).

[0229] In some embodiments, the Act IL-18 polypeptide exhibits a half-maximal effective concentration (EC50) for IL-18 receptor signaling activity (e.g., in a HEK-Blue reporter assay) which is higher than that of the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL-18 polypeptide exhibits an EC50 for IL-18 receptor signaling activity which is at least 1,000-fold higher, 2,000-fold higher, 5,000-fold higher, 10,000-fold higher, 15,000-fold-higher, or 20,000-fold higher than the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL- 18 polypeptide exhibits an EC50 for IL- 18 receptor signaling activity which is at least 1,000-fold higher than the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL-18 polypeptide exhibits an EC50 for IL-18 receptor signaling activity which is at least 5,000- fold higher than the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL- 18 polypeptide exhibits an EC50 for IL- 18 receptor signaling activity which is at least 10,000- fold higher than the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL- 18 polypeptide exhibits an EC50 for IL- 18 receptor signaling activity which is at least 20,000- fold higher than the activated form of the IL- 18 polypeptide.

[0230] In some embodiments, the Act-IL-18 polypeptide exhibits only a modest reduction in activity compared to the activated form of the IL- 18 polypeptide. In some embodiments, the Act IL- 18 polypeptide exhibits a half-maximal effective concentration (EC50) for IL- 18 receptorWSGR Docket No. 56146-747.601 signaling activity which is from about 10-fold higher to about 100-fold higher than the activated form of the IL-18 polypeptide. In some embodiments, the Act IL-18 polypeptide exhibits an EC50 for IL-18 receptor signaling activity which is from about 10-fold higher to about 50-fold higher than the activated form of the IL- 18 polypeptide.

[0231] In some embodiments, the activated form of the IL-18 polypeptide has a comparable activity compared that of the IL-18 polypeptide from which the Act-IL-18 polypeptide is derived. In some embodiments, the activated form of the IL- 18 polypeptide exhibits a half-maximal effective concentration (EC50) for IL- 18 receptor signaling activity which is within about 10-fold of the IL- 18 polypeptide.Exemplary Act-IL- 18 polypeptides and Corresponding Controls

[0232] Table 4 below provides exemplary Act-IL- 18 polypeptides according to the instant disclosure and corresponding controls. The full sequence of the Act-IL-18 polypeptides provided in the table can be arrived at by combining, in an N-terminal to C-terminal direction, the blocking moiety sequence, the cleavage and / or linker sequence, and the IL-18 polypeptide with the indicated modifications, wherein the modifications are relative to the mature human wild type IL- 18 sequence of SEQ ID NO: 701, except for compositions C172, C173, C189, C191, C136, and C137, which full length sequences can be arrived at by combining, in an N-terminal to C-terminal direction, the IL- 18 polypeptide with the indicated modifications, the cleavage and / or linker sequence, and the blocking moiety sequence.TABLE 7: EXEMPLARY ACT-IL-18 SEQUENCES AND CONTROLSWSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601*Cleavable peptide sequence denoted with an * form an additional cleavage site with the N- terminal YFG of the IL-18 polypeptide (where present).In some embodiments, an activatable IL-18 polypeptide is one described in Table 4.Conjugation Vs. Fusion of IL-18 Polypeptide in Immunocytokine Compositions

[0233] In some embodiments, an IL-18 polypeptide as described herein is attached to the immunocytokine composition via conjugation (i.e., by a conjugation reaction, such as with a conjugation handle). In some embodiments, an IL-18 polypeptide as described herein is fused to another portion of the immunocytokine composition. In some embodiments, the IL- 18 polypeptideWSGR Docket No. 56146-747.601 is fused via its C-terminus, optionally via a peptide linker. In some embodiments, the IL-18 polypeptide is fused via its N-terminus, optionally via a peptide linker.Scaffolds for Multifunctional Immunocytokine Compositions

[0234] In some embodiments, the components of the immunocytokine composition (e.g., the anti- PD-1 binding domain, the anti-VEGFA binding domain, and the cytokine (e.g., the IL-18 polypeptide)) are all in covalent association. In some embodiments, each component is linked to the other portions of the immunocytokine composition via covalent bonds. In some embodiments, the portions of the immunocytokine composition are all linked to a scaffold group. Non -limiting examples of suitable scaffold groups include, for example, polypeptides (e.g., immunoglobulins or other biocompatible polypeptides), polymers (e.g., biocompatible polymers), particles (e.g., nanoparticles, microparticles, etc., such as those made from biocompatible polymers or metals), or any other such groups. In some preferred embodiments, all of the components are linked to a scaffold polypeptide, such as an immunoglobulin polypeptide. In some embodiments, one or more of the portions of the immunocytokine composition can be linked to the scaffold indirectly, such as through linkers (e.g., any of the linkers described herein, including peptide linkers for fusion proteins) or through other portions of the immunocytokine composition (e.g., the two binding domains are linked such that only one of the binding domains is attached to the scaffold (optionally through a suitable linker) and the other binding domain is linked the binding domain which is linked to the scaffold, independently and optionally though another linker). In some embodiments, all of the components are linked as a fusion protein of one or more polypeptides which combine to form the immunocytokine composition (e.g., each component is fused, directly or indirectly, to a polypeptide scaffold, such as an Fc domain).Fc Domains as Scaffolds

[0235] In some embodiments, the immunocytokine composition comprises an Fc domain which acts as a scaffold. In some embodiments, the Fc domain is fused to one, both, or each of the binding domains (or, for binding domains which comprise a plurality of polypeptide chains, such as a Fab, one of the polypeptide chains of the binding domain). In some embodiments, the Fc domain is also fused to the cytokine (e.g., the IL- 18 polypeptide).

[0236] In some embodiments, the Fc domain is conjugated to the cytokine (e.g., the IL-18 polypeptide) via a linker. In some embodiments, such a linker is attached to a side chain of an amino acid residue of the Fc domain (e.g., a K246, K248, K288, K290, or K317 residue of the Fc domain (EU numbering), such as by AJICAP™ technology). Any of the Fc domains described herein (e.g., any one of SEQ ID NOs: 229-234 or 236-241, or a variant thereof) can comprise such a linker attached. In some embodiments, one arm of an Fc domain comprises a modification which prevents attachment of a linker at a specific residue, thereby facilitating conjugation of only aWSGR Docket No. 56146-747.601 single group to the Fc domain scaffold (e.g., a substitution at one of residues K246, K248, K288, K290, or K317 on one arm of the Fc domain, thereby preventing attachment of the linker to that arm when using AJICAP™ technology). In some embodiments, the Fc domain comprises a K246A, K248A, K288A, K290A, or K317A substitution. In some embodiments, the Fc domain comprises a K248A substitution. Any of the Fc domains described herein (e.g., any one of SEQ ID NOs: 229-234 or 236-241, or a variant thereof) can comprise such a modification.

[0237] In some embodiments, an immunocytokine composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain.

[0238] In some embodiments, the immunocytokine composition comprises an Fc domain. In some embodiments, the Fc domain is an IgG Fc domain, an IgA Fc domain, an IgD Fc domain, an IgM Fc domain, or an IgE Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG Fc domain, an IgA Fc domain, or an IgD Fc domain, or a derivative thereof. In some embodiments, the Fc domain is a human Fc domain, or a derivative thereof. In some embodiments, the Fc domain is a humanized. Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG Fc domain, or a derivative thereof. In some instances, an IgG Fc domain is an IgGl Fc domain, an IgG2a Fc domain, or an IgG4 Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgGl Fc domain, or a derivative thereof. In some embodiments, the Fc domain is an IgG4 Fc domain, or a derivative thereof. In some embodiments, a derivative of an Fc domain is one which contains at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a corresponding natural Fc domain (e.g., each arm has the indicated sequence identity).

[0239] In some embodiments, the Fc domain of an immunocytokine composition is an IgGl Fc domain, or a derivative thereof. In some embodiments, the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a human IgGl Fc domain (i.e., the CH2 and CH3 domains of human IgGl) (e.g., each arm of the Fc domain has the indicated sequence identity). In some embodiments, the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in the table below. In some embodiments, the Fc domain which comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in the table below can further comprise residues corresponding to residues 231-238 of an IgGl (EU numbering) appended to the N-terminal residue of the sequences set forth in the table below (e.g., a peptide sequence of APELLGGP (SEQ ID NO: 227) (WT IgGl “lower hinge”) or APEAAGGP (SEQ ID NO: 228) (LALA substituted “lower hinge”)). Such a sequence can also be considered to be co-extensive with theWSGR Docket No. 56146-747.601 hinge region. Thus, when a dual binding composition described herein is described as having a hinge region described herein and a Fc domain as described herein, it is contemplated that the sequence of the hinge region described herein and the Fc domain described herein can contain overlap. In some embodiments, the Fc domain with the indicated sequence identity to the sequence set forth in the table below retains the noted substitutions (all of which are numbered with EU numbering). In some embodiments, the Fc domain comprises one or more additional substitutions described herein (e.g., K248A on one arm, etc.). In some embodiments, the Fc domain comprises one or more additional substitutions described herein (e.g., K248A on one arm, etc.) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more additional substitutions).Table 8 - Fc Domain SequencesWSGR Docket No. 56146-747.601

[0240] In some embodiments, the Fc domain of an immunocytokine composition is an IgG4 Fc domain, or a derivative thereof. In some embodiments, the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a human IgG4 Fc domain (i.e., the CH2 and CH3 domains of human IgG4) (e.g., each arm of the Fc domain has the indicated sequence identity). In some embodiments, the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in the table below. In some embodiments, the Fc domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence set forth in the table below in which the first 8 residues of the sequences set forth below are omitted (e.g., the sequence APEFLGGP (SEQ ID NO: 235) is omitted). Such a sequence can also be considered to be co-extensive with the hinge region. Thus, when a dual binding composition described herein is described as having a hinge region described herein and a Fc domain as described herein, it is contemplated that the sequence of the hinge region described herein and the Fc domain described herein can contain overlap (i.e., when a dual binding composition is described as having an Fc domain described herein and a hinge region described herein, any overlapping portions of such sequences can be attributed to both regions). In some embodiments, the Fc domain with the indicated sequence identity to the sequence set forth in the table below retains the noted substitutions (all of which are numbered with EU numbering). In some embodiments, the Fc domain comprises one or more additional substitutionsWSGR Docket No. 56146-747.601 described herein (e.g., K248A on one arm, etc.) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more additional substitutions).Table 9 -Additional Fc Domain SequencesWSGR Docket No. 56146-747.601

[0241] One or more mutations may be introduced in an Fc domain to reduce Fc-mediated effector functions of immunocytokine composition, such as, for example, antibody-dependent cellular cytotoxicity (ADCC) and / or complement function. In some instances, a modified Fc comprises a humanized IgG4 kappa isotype that contains a S229P Fc mutation. In some instances, a modified Fc comprises a human IgGl kappa where the heavy chain CH2 domain is engineered with a triple mutation such as, for example: (a) L238P, L239E, and P335S; or (2) K248; K288; and K317.

[0242] In some embodiments, the Fc domain comprises one or more modifications which favors heterodimerization of the two polypeptide which dimerize to form the Fc domains. Many such modifications are known in art for generating bispecific antibodies which can be applied to the instant disclosure. Such modifications are described in, for example, “Fc Engineering for Developing Therapeutic Bispecific Antibodies and Novel Scaffolds,” Liu et al., Front. Immunol., 26 January 2017 (doi.org / 10.3389 / fininiu.2017.00038) and include, for example, knob-into-hole technology (see, e.g., U.S. Patent No. 8,216,805) and modification introduced into one Fc domain to abrogate binding to protein A to facilitate purification of desired heterodimeric formats {e.g., RF mutations, as described in, e.g., U.S. Patent No. 11,168,111). In some embodiments, the Fc domain of the immunocytokine compositions provided herein utilize knob-into-hole technology, for example the “hole” modifications of Y349C, T366S, L368A, and Y407V and the “knob” modifications of S354C and T366W (EU numbering). In some embodiments, the immunocytokine compositions provided herein utilize the RF mutations, e.g., H435R and Y436F mutations (EU numbering). In some embodiments, the immunocytokine compositions utilize both of these modifications together {e.g., one arm of the Fc domain of the immunocytokine composition having the hole and RF modifications, and one arm of the Fc domain of the immunocytokine composition having the knob modifications). In some embodiments, on arm of the Fc domain comprises T336W, H435R, and Y436F substitutions and the other arm of the Fc domain comprises T366S, L368A, Y407V substitutions. In some embodiments, one arm of the Fc domain comprises Y349C, T366S, L368A, and Y407V “hole” mutations and the other arm of the Fc domain comprises S354C and T366W “knob” mutations (EU numbering). Other combinations of such knob and hole modifications are well known in the art and compatible with the instant disclosure.

[0243] In some embodiments, one arm of the Fc domain of the immunocytokine composition comprises a mutation at a residue which eliminates the ability to conjugate an additional groupWSGR Docket No. 56146-747.601(such as a cytokine, in particular an IL- 18 polypeptide as described herein) to that site, thereby allowing for easier preparation of an immunocytokine composition which contains only a single cytokine (e.g., only one IL-18 polypeptide as described herein). For example, in instances where AJICAP™ technology is intended to be used to conjugate the cytokine (e.g., the IL-18 polypeptide) to one of residues K246, K248, K288, K290, or K317 (EU numbering), one arm of the Fc domain can comprise a mutation at the lysine residue to be targeted for conjugation to render it unavailable for reaction with the affinity peptide of the AJICAP™ technology. In one particular example, where it is intended to use an AJICAP™ affinity peptide to add a sulfide group to K248 for subsequent conjugation to the cytokine (e.g., through an intermediate reaction with a heterobifunctional linking reagent described herein), the K248 residue of one arm of the Fc domain is mutated to a suitable residue which is incapable of reaction with the affinity peptide, such as a K248A substitution. Thus, in some embodiments, one arm of the Fc domain contains a substitution at one or more of residue K246, K248, K288, K290, or K317. In some embodiments, the substitution is one or more of a K246A, K248A, K288A, K290A, or K317A substitution. In some embodiments, one arm of the Fc domain contains a K248A substitution. In some embodiments, one arm of the Fc domain contains a K248A substitution and the other arm is conjugated to the cytokine (e.g., the IL-18 polypeptide). Analogously, mutations at the other residues on one arm of the Fc domain can similarly be paired with conjugation at the corresponding unsubstituted residue on the other arm of the Fc domain (e.g., one arm with a K246A substitution and the other arm conjugated to the cytokine). Such substitutions to facilitate conjugation to only one arm of the Fc domain are favorably paired with substitutions which help to facilitate the heterodimerization of the arms of the Fc domain (e.g., knob-into-hole modifications discussed above or other similar modifications known in the art).

[0244] In some embodiments, the constant domains (e.g., the Fc domain) of an immunocytokine composition described herein can comprise further modifications (either in place of or in addition to the other modifications described herein, such as those which favor heterodimerization of two different arms of the fusion immunocytokine). Such modifications to Fc domains are known in the art and include, for example, modifications which alter antibody effector functions (e.g., enhance or decease Fc receptor binding or activity, thereby altering antibody-dependent cellular cytotoxicity, complement dependent cytotoxicity, or other effects), improve half-life circulation, or otherwise alter the performance of the molecule. Such modifications are well known in the art and are described in, for example, “Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life,” Saunders et al., Fron. Immunol., 07 June 2019 (doi.org / 10.3389 / fimmu.2019.01296). Such modifications can be at any relevant portion of the fusion immunocytokine, including without limitation an Fc domain (e.g., either the CH2 or CH3WSGR Docket No. 56146-747.601 domain, or both), a hinge region, a CHI domain, a light chain constant region, and / or a framework region of an antigen binding domain (e.g., a VH or VL domain).Conjugation to Fc domains

[0245] In some embodiments, the immunocytokine compositions described herein comprise an Fc domain, wherein the Fc domain comprises at least one covalently linked linker to a side chain of the Fc domain. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is covalently attached to a tyrosine, aspartic acid, glutamic acid, arginine, histidine, or lysine residue. In some embodiments, the chemical linker is covalently attached to a lysine, cysteine, or tyrosine residue. In some embodiments, the chemical linker is covalently attached to a cysteine residue. In some embodiments, the chemical linker is covalently attached to a lysine residue. In some embodiments, such a linker attaches the cytokine (e.g., the IL- 18 polypeptide) to the Fc domain.

[0246] In some embodiments, the immunocytokine composition comprises one or both of the anti- PD-1 and / or the anti-VEGF binding domains fused to an Fc scaffold to which the cytokine (e.g., the IL-18 polypeptide) can be added by conjugation. Non-limiting examples of Fc domain containing scaffolds fused to anti-PD-1 and / or anti-VEGF binding domains suitable for addition of the cytokine (e.g., the IL-18 polypeptide) to form the immunocytokine composition are depicted in FIGs. 2A-D, 3A-D, 4A-C, and 5A-B. Non-limiting examples of sequences of such Fc domain scaffolds fused to anti-VEGF and anti-PD-1 binding domains can be found in the table below. In some embodiments, a cytokine of the instant disclosure (e.g., an IL-18 polypeptide described herein) is conjugated to one of the Fc-domain containing polypeptides below, such as by use of AJICAP™ technology to add a sulfide conjugation handle to a K246, K248, K288, K290, or K317 residue of the Fc domain (EU numbering). In some embodiments, a cytokine of the instant disclosure (e.g., an IL-18 polypeptide described herein) is fused to one of the Fc-domain containing polypeptides below, such by, for example, by replacing a VH and CHI domain from a sequence below with the cytokine.TABLE 10 - Exemplary Constructs Targeting VEGFA and PD-1WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601WSGR Docket No. 56146-747.601

[0247] The chemical linker can be covalently attached to one amino acid residue of an Fc region of the immunocytokine composition. In some embodiments, the chemical linker is covalently attached to a non -terminal residue of the Fc region. In some embodiments, the non -terminal residue is in the CHI, CH2, or CH3 region of the immunocytokine composition. In some embodiments, the non-terminal residue is in the CH2 region of the immunocytokine composition.

[0248] In some embodiments, the chemical linker is covalently attached at an amino acid residue of the immunocytokine composition such that the function of a binding domain fused to the Fc domain is maintained (e.g., without denaturing the polypeptide). For example, when immunocytokine composition comprises a human IgG (e.g., human IgGl), exposed lysine residues and exposed tyrosine residues are present at the following positions (refer to web site www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html by EU numbering). Exemplary exposed Lysine Residues: CH2 domain (position 246, position 248, position 274, position 288, position 290, position 317, position 320, position 322, and position 338) CH3 domain (position 360, position 414, and position 439). Exemplary exposed Tyrosine Residues: CH2 domain (position 278, position 296, and position 300) CH3 domain (position 436).

[0249] The human IgG, such as human IgGl, may also be modified with a lysine or tyrosine residue at any one of the positions listed above in order provide a residue which is ideally surface exposed for subsequent modification.

[0250] In some embodiments, the chemical linker is covalently attached at an amino acid residue in the constant region of an immunocytokine composition. In some embodiments, the chemicalWSGR Docket No. 56146-747.601 linker is covalently attached at an amino acid residue in the CHI, CH2, or CH3 region. In some embodiments, the chemical linker is covalently attached at an amino acid residue in the CH2 region. In some embodiments, the chemical linker may be covalently attached to one amino acid residue in the following groups of residues following EU numbering in human IgG Fc: amino acid residues 1-478, amino acid residues 2-478, amino acid residues 1-477, amino acid residues 2-477, amino acid residues 10-467, amino acid residues 30-447, amino acid residues 50-427, amino acid residues 100-377, amino acid residues 150-327, amino acid residues 200-327, amino acid residues 240-327, and amino acid residues 240-320.

[0251] In some embodiments, the chemical linker is covalently attached to one lysine residue of a human IgG Fc domain (e.g. IgGl or IgG4). In some embodiments, the chemical linker is covalently attached at Lys 246, Lys 248, Lys 288, Lys 290, or Lys 317 of the Fc domain (EU numbering). In some embodiments, the chemical linker is covalently attached at Lys 246 of an Fc region of the Fc domain, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 248 of an Fc domain of the immunocytokine composition, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 288 of an Fc region of the immunocytokine composition, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 290 of an Fc region of the immunocytokine composition, wherein amino acid residue position number is based on EU numbering. In some embodiments, the chemical linker is covalently attached at Lys 317 of the immunocytokine composition, wherein amino acid residue position number is based on EU numbering.

[0252] The chemical linker can be covalently attached to an amino acid residue selected from a subset of amino acid residues. In some embodiments, the subset comprises two three, four, five, six, seven, eight, nine, or ten amino acid residues of an Fc region of the immunocytokine composition. The chemical linker can be covalently attached to one of two lysine residues of an Fc region of the immunocytokine composition.

[0253] In some embodiments, the Fc domain will comprise two linkers covalently attached to the Fc region of the immunocytokine composition. In some embodiments, each of the two linkers will be covalently attached to a different heavy chain of the immunocytokine composition. However, in preferred embodiments of immunocytokine compositions described herein, only a single linker is attached to the Fc domain or antigen binding fragment thereof, thereby providing an immunocytokine composition which includes only a single IL- 18 polypeptide described herein.WSGR Docket No. 56146-747.601Methods For Attaching Linkers to Fc Domains

[0254] Also provided herein are method of preparing a modified Fc region of an immunocytokine composition, such as for the attachment of a linker, a conjugation handle, the cytokine (e.g., the IL-18 polypeptide), or any combination thereof to the Fc domain to form the immunocytokine composition. A variety of methods for site-specific modification of Fc regions are known in the art.Modification with an affinity peptide configured to site-specifically attach linker to the Fc domain

[0255] In some embodiments, an Fc region is modified to incorporate a linker, a conjugation handle, or a combination thereof. In some embodiments, this is accomplished using AJICAP™ technology, or a similar technology known in the art. In some embodiments, the modification is performed by contacting the Fc region with an affinity peptide bearing a payload configured to attach a linker or other group to the Fc region, such as at a specific residue of the Fc region. In some embodiments, the linker is attached using a reactive group which forms a bond with a residue of the Fc region. In some embodiments, the affinity peptide comprises a cleavable linker. The cleavable linker is configured on the affinity peptide such that after the linker or other group is attached to the Fc region, the affinity peptide can be removed, leaving behind only the desired linker or other group attached to the Fc region. The linker or other group can then be used further to add attach additional groups, such as a cytokine or a linker attached to a cytokine, to the Fc region.

[0256] Non-limiting examples of such affinity peptides commensurate with AJICAP™ technology can be found at least in PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288 Al, PCT Publication No. WO2019240287A1, and PCT Publication No. W02020090979A1, each of which is incorporated by reference as if set forth herein in its entirety. In some embodiments, the affinity peptide is a peptide which has been modified to deliver the linker / conjugation handle payload one or more specific residues of the Fc region of the immunocytokine composition.

[0257] An exemplary affinity peptide with cleavable linker and conjugation handle payload capable of attaching the payload to residue K248 of Fc domain as provided can be found in in Matsuda et al., “Chemical Site-Specific Conjugation Platform to Improve the Pharmacokinetics and Therapeutic Index of Antibody-Drug Conjugates,” Mol. Pharmaceutics 2021, 18, 11, 4058- 4066.

[0258] Alternative affinity peptides targeting alternative residues of the Fc region are described in the references cited above for AJICAP™ technology, and such affinity peptides can be used to attach the desired functionality to an alternative residue of the Fc region (e.g., K246, K288, etc.). Such alternative affinity peptides include those described in, for example “AJICAP SecondWSGR Docket No. 56146-747.601Generation: Improved Chemical Site-Specific Conjugation Technology for Antibody-Drug Conjugation Technology for Antibody-Drug Conjugate Production” (Working Paper, Fujii et al., DOI: 10.26434 / chemrxiv-2023-9p5p7, chemrxiv.org / engage / chemrxiv / article- details / 63d5f7131125965a9e7df8a5 (Accessed 20 February 2023, Version 1 published 30 Jan 2023)).

[0259] The affinity peptide of the disclosure can comprise a cleavable linker. In some embodiments, the cleavable linker of the affinity peptide connects the affinity peptide to the group which is to be attached to the Fc region and is configured such that the peptide can be cleaved after the group comprising the linker or conjugation handle has been attached. In some embodiments, the cleavable linker is a divalent group. In some embodiments, the cleavable linker can comprise a thioester group, an ester group, a sulfane group; a methanimine group; an oxyvinyl group; a thiopropanoate group; an ethane- 1,2-diol group; an (imidazole- l-yl)methan-l -one group; a seleno ether group; a silylether group; a di-oxysilane group; an ether group; a di-oxymethane group; a tetraoxospiro[5.5]undecane group; an acetamidoethyl phosphorami di te group; a bis(methylthio)-pyrazolopyrazole-dione group; a 2-oxo-2-phenylethyl formate group; a 4- oxybenzylcarbamate group; a 2-(4-hydroxy-oxyphenyl)diazinyl)benzoic acid group; a 4-amino- 2-(2-amino-2-oxoethyl)-4-oxobut-2-enoic acid group; a 2-(2-methylenehydrazineyl)pyridine group; an N'-methyleneformohydrazide group; or an isopropylcarbamate group, any of which is unsubstituted or substituted. Composition and points of attachment of the cleavable linker to the affinity peptide, as well as related methods of use, are described in, at least, PCT Publication No. WO2018199337A1, PCT Publication No. WO2019240288 Al, PCT Publication No. WO2019240287A1, and PCT Publication No. W02020090979A1.

[0260] In some embodiments, the cleavable linker is:WSGR Docket No. 56146-747.601wherein:-one of A or B is a point of attachment the linker and the other of A or B is a point of attachment to the affinity peptide;- each R2ais independently H or optionally substituted alkyl;- each R2bis independently H or optionally substituted alkyl;- R2Cis a H or optionally substituted alkyl;-J is a methyl, a N, a S, a Si, or an O atom; and- r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0261] The affinity peptide comprises a reactive group which is configured to enable the covalent attachment of the linker / conjugation handle to the Fc region. In some embodiments, the reactive group is selective for a functional group of a specific amino acid residue, such as a lysine residue, tyrosine residue, serine residue, cysteine residue, or an unnatural amino acid residue of the Fc region incorporated to facilitate the attachment of the linker. The reactive group may be any suitable functional group, such as an activated ester for reaction with a lysine (e.g., N- hydroxy succinimide ester or a derivate thereof, a pentafluorophenyl ester, etc.) or a sulfhydryl reactive group for reaction with a cysteine (e.g., a Michael acceptor, such as an alpha-beta unsaturated carbonyl or a maleimide). In some embodiments, the reactive group is:WSGR Docket No. 56146-747.601, wherein:- each Rsa, Rsb, and Rscis independently H, halogen, or optionally substituted alkyl;- each j is 1, 2, 3, 4, or 5; and- each k is 1, 2, 3, 4, or 5.

[0262] In some embodiments, the affinity peptide is used to deliver a reactive moiety to the desired amino acid residue such that the reactive moiety is exposed upon cleavage of the cleavable linker. By way of non-limiting example, the reactive group forms a covalent bond with a desired residue of the Fc region of the immunocytokine composition due to an interaction between the affinity peptide and the Fc region. Following this covalent bond formation, the cleavable linker is cleaved under appropriate conditions to reveal a reactive moiety (e.g., if the cleavable linker comprises a thioester, a free sulfhydryl group is attached to the Fc region following cleavage of the cleavable linker). This new reactive moiety can then be used to subsequently add an additional moiety, such as a conjugation handle, by way of reagent comprising the conjugation handle tethered to a sulfhydryl reactive group (e.g., alpha-halogenated carbonyl group, alpha-beta unsaturated carbonyl group, mal eimide group, etc.).

[0263] In some embodiments, an affinity peptide is used to deliver a free sulfhydryl group to a lysine of the Fc region. In some embodiments, the free sulhydryl group added to the lysine of theFc region has a structure0, wherein the nitrogen atom shown is the side chain amine of the lysine. Preferably wherein there are 2 or 3 methylenes between the carbonyl and the sulfhydryl group of the structure shown. In some embodiments, the free sulfhydryl group is then reacted with a bifunctional linking reagent to attach a new conjugation handle to the Fc region. InWSGR Docket No. 56146-747.601 some embodiments, the new conjugation handle is then used to form the linker to the attached cytokine. In some embodiments, the new conjugation handle is an alkyne functional group. In some embodiments, the new conjugation handle is a DBCO functional group.

[0264] Exemplary bifunctional linking reagents useful for this purpose are of a formula A-B-C, wherein A is the sulfhydryl reactive conjugation handle (e.g., maleimide, a,P-unsaturated carbonyl, a-halogenated carbonyl), B is a linking group, and C is the new conjugation handle (e.g., an alkyne such as DBCO). Specific non-limiting examples of bifunctional linking reagents includen is independently an integer from 1-6 and each m is independently an integer from 1-30, and related molecules (e.g., isomers). In such examples, the DBCO group is reacted with an azide group attached to cytokine (e.g. the IL-18 polypeptide (e.g., at any of the residues provided herein, such as C68)).

[0265] In some embodiments of immunocytokines described herein, the bifunctional linking reagent used has the structure

[0266] Alternatively, the affinity peptide can be configured such that a conjugation handle is added to the Fc region (such as by a linker group) immediately after covalent bond formation between the reactive group and a residue of the Fc region. In such cases, the affinity peptide isWSGR Docket No. 56146-747.601 cleaved and the conjugation handle is immediately ready for subsequent conjugation to the cytokine (e.g., the IL-18 polypeptide).Alternative Methods of Modifying Fc Region

[0267] While the affinity peptide mediated modification of an Fc region provided supra possesses many advantages over other methods which can be used to site-specifically modify the Fc region (e.g., ease of use, ability to rapidly generate many different conjugates, ability to use many “off- the-shelf’ commercial antibodies without the need to do time consuming protein engineering, etc.), other methods of performing the modification are also contemplated as being within the scope of the present disclosure.

[0268] In some embodiments, a cytokine (e.g., an IL-18 polypeptide) can be conjugated to a suitable Fc Domain utilizing transglutaminase-mediated site-specific antibody-drug conjugate (ADC) strategies, such as those comprising: 1) glutamine-containing tags, endogenous glutamines (e.g., native glutamines without engineering, such as glutamines in variable domains, CDRs, etc.), and / or endogenous glutamines made reactive by antibody engineering or an engineered transglutaminase; and 2) amine donor agents comprising amine donor units, linkers, and agent moieties. Non-limiting examples of such transglutaminase mediated site-specific modifications can be found at least in publications PCT Publication No. W02020188061, US Patent Publication No. US2019194641, US Patent Publication No. US2021128743, US Patent No. US9764038, and US Patent No. US10434180, which are incorporated by reference as if set forth herein in their entirety. Such strategies can be employed to either add a suitable conjugation handle to a desired site of the Fc domain (or other part of the immunocytokine composition), or can alternatively be used to directly conjugate the cytokine (e.g., the IL-18 polypeptide) to the Fc domain, though it is preferable that such a strategy is used to add a conjugation handle, then the cytokine (e.g. IL-18 polypeptide) is later reacted with the conjugation handle to form the immunocytokine.

[0269] In some embodiments, a cytokine (e.g. IL-18 polypeptide) can be conjugated to a suitable Fc domain utilizing a transpeptide-mediated strategy for either direct attachment of the cytokine (e.g., IL-18 polypeptide) to the immunocytokine composition or via addition of a suitable conjugation handle to the Fc domain. For example, a sortase based system (such as those described in, for example, U.S. Patent No. 10,081,684, U.S. Patent No. 10,864,277, U.S. Patent No. 11,421,022, and / or U.S. Patent No. 9,862,779) can be used, either by addition of a sortase tag to the C-terminus of the Fc domain or through a suitable linker armed with a sortase tag, to add a linker with a conjugation handle to the Fc domain, followed by a subsequent conjugation of the cytokine to the immunocytokine composition using the conjugation handle.

[0270] In another aspect, the disclosure provides methods of generating immunocytokines using an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containingWSGR Docket No. 56146-747.601 polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine- containing tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., X can be the same or different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme).

[0271] In some embodiments, the acyl donor glutamine-containing tag is not spatially adjacent to a reactive Lys (e.g., the ability to form a covalent bond as an amine donor in the presence of an acyl donor and a transglutaminase) in the polypeptide or the Fc-containing polypeptide. In some embodiments, the polypeptide or the Fc-containing polypeptide comprises an amino acid modification at the last amino acid position in the carboxyl terminus relative to a wild-type polypeptide at the same position. The amino acid modification can be an amino acid deletion, insertion, substitution, mutation, or any combination thereof.

[0272] In some embodiments, the immunocytokine composition comprises a full length antibody heavy chain and an antibody light chain, wherein the acyl donor glutamine-containing tag is located at the carboxyl terminus of a heavy chain, a light chain, or both the heavy chain and the light chain.

[0273] In some embodiments, the immunocytokine composition comprises an antibody, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a bispecific antibody, a minibody, a diabody, or an antibody fragment. In some embodiments, the antibody is an IgG.

[0274] In another aspect, provided herein is a method for preparing an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc-containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine- containing tag at a carboxyl terminus, an amino terminus, or at an another site in the Fc-containing polypeptide, wherein the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., X can be the same or a different amino acid), and wherein the engineered Fc-containing polypeptide conjugate comprises an amino acid substitution from glutamine to asparagine at position 295 (Q295N; EU numbering scheme), comprising the steps of a) providing an engineered (Fc-containing polypeptide)-T molecule comprising the Fc- containing polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered (Fc-containing polypeptide)-T molecule in the presence of aWSGR Docket No. 56146-747.601 transglutaminase; and c) allowing the engineered (Fc-containing polypeptide)-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

[0275] In another aspect, provided herein is a method for preparing an engineered polypeptide conjugate comprising the formula: polypeptide-T-A, wherein T is an acyl donor glutamine- containing tag engineered at a specific site, wherein A is an amine donor agent, wherein the amine donor agent is site-specifically conjugated to the acyl donor glutamine-containing tag at a carboxyl terminus, an amino terminus, or at an another site in the polypeptide, and wherein the acyl donor glutamine-containing tag comprises an amino acid sequence GGLLQGPP (SEQ ID NO: 299), comprising the steps of: a) providing an engineered polypeptide-T molecule comprising the polypeptide and the acyl donor glutamine-containing tag; b) contacting the amine donor agent with the engineered polypeptide-T molecule in the presence of a transglutaminase; and c) allowing the engineered polypeptide-T to covalently link to the amine donor agent to form the engineered Fc-containing polypeptide conjugate.

[0276] In some embodiments, the engineered polypeptide conjugate (e.g., the engineered Fc- containing polypeptide conjugate, the engineered Fab -containing polypeptide conjugate, or the engineered antibody conjugate) as described herein has conjugation efficiency of at least about 51%. In another aspect, the invention provides a pharmaceutical composition comprising the engineered polypeptide conjugate as described herein (e.g., the engineered Fc-containing polypeptide conjugate, the engineered Fab -containing polypeptide conjugate, or the engineered antibody conjugate) and a pharmaceutically acceptable excipient.

[0277] In some embodiments, provided herein is a method for conjugating a moiety of interest (Z) to an Fc domain, comprising the steps of: (a) providing an Fc domain having (e.g., within the primary sequence of a constant region) at least one acceptor amino acid residue (e.g., a naturally occurring amino acid) that is reactive with a linking reagent (linker) in the presence of a coupling enzyme, e.g., a transamidase; and (b) reacting said Fc domain with a linking reagent (e.g., a linker comprising a primary amine) comprising a reactive group (R), optionally a protected reactive group or optionally an unprotected reactive group, in the presence of an enzyme capable of causing the formation of a covalent bond between the acceptor amino acid residue and the linking reagent (other than at the R moiety), under conditions sufficient to obtain an Fc domain comprising an acceptor amino acid residue linked (covalently) to a reactive group (R) via the linking reagent. Optionally, said acceptor residue of the Fc domain or Fc domain fragment is flanked at the +2 position by a non-aspartic acid residue. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, nonglutamine residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, nonasparagine residue. In one embodiment, the residue at the +2 position is a non-negatively chargedWSGR Docket No. 56146-747.601 amino acid (an amino acid other than an aspartic acid or a glutamic acid). Optionally, the acceptor glutamine is in an Fc domain optionally further- within the CH2 domain Optionally, the Fc domain is free of heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295 and the residue at the +2 position is the residue at position 297 (EU index numbering) of an Fc domain.

[0278] In one aspect, provided herein is a method for conjugating a moiety of interest (Z) to an Fc domain, comprising the steps of: (a) providing an Fc domain having at least one acceptor glutamine residue; and (b) reacting said Fc domain with a linker comprising a primary amine (a lysine-based linker) comprising a reactive group (R), preferably a protected reactive group, in the presence of a transglutaminase (TGase), under conditions sufficient to obtain an Fc domain comprising an acceptor glutamine linked (covalently) to a reactive group (R) via said linker. Optionally, said acceptor glutamine residue of the Fc domain is flanked at the +2 position by a non-aspartic acid residue. Optionally, the residue at the +2 position is a non-aspartic acid residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-glutamine residue. In one embodiment, the residue at the +2 position is a non-aspartic acid, non-asparagine residue. In one embodiment, the residue at the +2 position is a non-negatively charged amino acid (an amino acid other than an aspartic acid or a glutamic acid). Optionally, the acceptor glutamine is in an Fc domain of an Fc domain, optionally further-within the CH2 domain Optionally, the Fc domain is free of heavy chain N297-linked glycosylation. Optionally, the acceptor glutamine is at position 295 and the residue at the +2 position is the residue at position 297 (EU index numbering) of an Fc domain. The Fc domain comprising an acceptor residue or acceptor glutamine residue linked to a reactive group (R) via a linker comprising a primary amine (a lysine-based linker) can thereafter be reacted with a reaction partner comprising a moiety of interest (Z) to generate an Fc domain comprising an acceptor residue or acceptor glutamine residue linked to a moiety of interest (Z) via the linker. Thus, in one embodiment, the method further comprises a step (c): reacting (i) an Fc domain of step b) comprising an acceptor glutamine linked to a reactive group (R) via a linker comprising a primary amine (a lysine-based linker), optionally immobilized on a solid support, with (ii) a compound comprising a moiety of interest (Z) and a reactive group (R1) capable of reacting with reactive group R, under conditions sufficient to obtain an Fc domain comprising an acceptor glutamine linked to a moiety of interest (Z) via a linker comprising a primary amine (a lysine-based linker). Preferably, said compound comprising a moiety of interest (Z) and a reactive group (R1) capable of reacting with reactive group R is provided at a less than 80 times, 40 times, 20 times, 10 times, 5 times or 4 molar equivalents to the Fc domain. In one embodiment, the Fc domain comprises two acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 10 or less molar equivalents to the Fc domain.WSGR Docket No. 56146-747.601In one embodiment, the Fc domain comprises two acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 5 or less molar equivalents to the Fc domain. In one embodiment, the Fc domain comprises four acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 20 or less molar equivalents to the Fc domain. In one embodiment, the Fc domain comprises four acceptor glutamines and the compound comprising a moiety of interest (Z) and a reactive group (R1) is provided at 10 or less molar equivalents to the Fc domain. In one embodiment, steps (b) and / or (c) are carried out in aqueous conditions. Optionally, step (c) comprises: immobilizing a sample of an Fc domain comprising a functionalized acceptor glutamine residue of Formula II on a solid support to provide a sample comprising immobilized antibodies, reacting the sample comprising immobilized antibodies, optionally recovering any unreacted compound and re-introducing such recovered compound to the solid support for reaction with immobilized antibodies, and eluting the Fc domain conjugates to provide an Fc domain composition comprising a Z moiety.

[0279] In an alternative embodiment, an amino acid residue comprising a conjugation handle can be incorporated into the Fc domain (e.g., during expression of the Fc domain) at a desired location (e.g., any of the locations provided herein). In some embodiments, the amino acid residue comprising the conjugation handle is an unnatural amino acid.Conjugation Handle Chemistry

[0280] In some embodiments, the appropriately modified Fc region of the immunocytokine composition will comprise a conjugation handle which is used to conjugate the immunocytokine composition to a cytokine (e.g. IL-18 polypeptide) to produce an immunocytokine composition provided herein.

[0281] Any suitable reactive group capable of reacting with a complementary reactive group attached to the cytokine can be used as the conjugation handle. In some embodiments, the conjugation handle comprises a reagent for a Cu(I) -catalyzed or "copper-free" alkyne-azide triazole-forming reaction (e.g., strain promoted cycloadditions), the Staudinger ligation, inverseelectron-demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, tetrazine cycloadditions with trans-cyclooctenes, potassium acyl trifluoroborate (KAT) ligation or a metal- mediated process such as olefin metathesis and Suzuki- Miyaura or Sonogashira cross-coupling.

[0282] In some embodiments, the conjugation handle comprises a reagent for a “copper-free” alkyne azide triazole-forming reaction. Non-limiting examples of alkynes for said alkyne, azide triazole forming reaction include cyclooctyne reagents (e.g., (lR,8S,9s)-Bicyclo[6.1.0]non-4-yn- 9-ylmethanol containing reagents, dibenzocyclooctyne-amine reagents, difluorocyclooctynes, orWSGR Docket No. 56146-747.601 derivatives thereof). In some embodiments, the alkyne functional group is attached to the Fc region. In some embodiments, the azide functional group is attached to the Fc region.

[0283] In some embodiments, the conjugation handle comprises a reactive group selected from azide, alkyne, tetrazine, halide, sulfhydryl, disulfide, maleimide, activated ester, alkene, aldehyde, ketone, imine, hydrazine, potassium acyl trifluorob orate, hydroxylamine (e.g., O-substituted hydroxylamine) and hydrazide. In some embodiments, the cytokine comprises a reactive group complementary to the conjugation handle of the Fc region. In some embodiments, the conjugation handle and the complementary conjugation handle comprise “CLICK” chemistry reagents. Exemplary groups of click chemistry residue are shown in Hein et aL, “Click Chemistry, A Powerful Tool for Pharmaceutical Sciences,” Pharmaceutical Research, volume 25, pages 2216- 2230 (2008); Thirumurugan et aL, “Click Chemistry for Drug Development and Diverse Chemical-Biology Applications,” Chem. Rev. 2013, 113, 7, 4905-4979; US20160107999A1; US10266502B2; and US20190204330A1, each of which is incorporated by reference in its entirety.Linker Structure

[0284] In some embodiments, the linker used to attach the Fc domain and the cytokine (e.g., IL- 18 polypeptide) comprises points of attachment at both moi eties. The points of attachment can be any of the residues for facilitating the attachment as provided herein. The linker structure can be any suitable structure for creating the spatial attachment between the two moieties. In some embodiments, the linker provides covalent attachment of both moieties. In some embodiments, the linker is a chemical linker (e.g., not an expressed polypeptide as in a fusion protein). In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a non-peptide linker (e.g., does not consist of amino acid residues).Chemical Linkers

[0285] In some embodiments, the linker is a chemical linker. Chemical linkers are generally used in the context of chemically conjugated multifunctional immunocytokines (e.g., immunocytokines in which the cytokine is conjugated to a scaffold comprising the binding domains specific for PD- 1 and VEGFA). In some embodiments, the chemical linker comprises at least one portion which is not comprised of amino acid residues. In some embodiments, the linker comprises a polymer. In some embodiments, the linker comprises a water soluble polymer. In some embodiments, the linker comprises poly(alkylene oxide), polysaccharide, poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the linker comprises poly(alkylene oxide). In some embodiments, the poly(alkylene oxide) is polyethylene glycol or polypropylene glycol, or a combination thereof. In some embodiments, the poly(alkylene oxide) is polyethylene glycol.WSGR Docket No. 56146-747.601

[0286] In some embodiments, the linker is a bifunctional linker. In some embodiments, the bifunctional linker comprises an amide group, an ester group, an ether group, a thioether group, or a carbonyl group. In some embodiments, the linker comprises a non-polymer linker. In some embodiments, the linker comprises a non-polymer, bifunctional linker. In some embodiments, the non-polymer, bifunctional linker comprises succinimidyl 4-(N-maleimidomethyl)cyclohexane-l- carboxylate; Maleimidocaproyl; Valine-citrulline; Allyl(4-methoxyphenyl)dimethylsilane; 6- (Allyloxycarbonylamino)-l-hexanol; 4-Aminobutyraldehyde diethyl acetal; or (E)-N-(2- Aminoethyl)-4-{2-[4-(3-azidopropoxy)phenyl]diazenyl}benzamide hydrochloride.

[0287] The linker can be branched or linear. In some embodiments, the linker is linear. In some embodiments, the linker is branched. In some embodiments, the linker comprises a linear portion (e.g., between the first point of attachment and the second point of attachment) of a chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker comprises a linear portion of a chain of at least 10, 20, 30, 40, or 50 atoms. In some embodiments, the linker comprises a linear portion of at least 10 atoms. In some embodiments, the linker is branched and comprises a linear portion of a chain of at least 10, 20, 50, 100, 500, 1000, 2000, 3000, or 5000 atoms. In some embodiments, the linker comprises a linear portion of at from 1 to 1000 atoms, 1 to 900 atoms, 1 to 800 atoms, 1 to 500 atoms, 1 to 400 atoms, 1 to 300 atoms, 1 to 200 atoms, 1 to 100 atoms, 1 to 50 atoms, 10 to 1000 atoms, 10 to 900 atoms, 10 to 800 atoms, 10 to 500 atoms, 10 to 400 atoms, 10 to 300 atoms, 10 to 200 atoms, 10 to 100 atoms, 10 to 50 atoms, 25 to 1000 atoms, 25 to 900 atoms, 25 to 800 atoms, 25 to 500 atoms, 25 to 400 atoms, 25 to 300 atoms, 25 to 200 atoms, 25 to 100 atoms, 25 to 50 atoms, 50 to 1000 atoms, 50 to 900 atoms, 50 to 800 atoms, 50 to 500 atoms, 50 to 400 atoms, 50 to 300 atoms, 50 to 200 atoms, or 50 to 100 atoms. In some embodiments, the linker has a linear length of from about 10 angstroms to about 200 angstroms. In some embodiments, the linker has a linear length of from about 10 to 500, 10 to 200, 10 to 150, 10 to 125, 10 to 100, 10 to 75, 10 to 50, 25 to 200, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 150, 50 to 100, or 50 to 75 angstroms.

[0288] In some embodiments, the linker has a molecular weight of about 200 Daltons to about 2000 Daltons. In some embodiments, the linker has a molecular weight of about 200 Daltons to about 5000 Daltons. In some embodiments, the linker has a molecular weight of 200 Daltons to 100,000 Daltons. In some embodiments, the linker has a molecular weight of at least about 500 Daltons, at least about 1,000 Daltons, at least about 5,000 Daltons, at least about 10,000 Daltons, at least about 15,000 Daltons, at least about 20,000 Daltons, at least about 25,000 Daltons, or at least about 30,000 Daltons. In some embodiments, the linker as a molecular weight of at most about 100,000 Daltons, at most about 50,000 Daltons, at most about 40,000 Daltons, at most aboutWSGR Docket No. 56146-747.60130,000 Daltons, at most about 25,000 Daltons, at most about 20,000 Daltons at most about 15,000 Daltons, at most about 10,000 Daltons, or at most about 5,000 Daltons.

[0289] In some embodiments, the linker comprises a reaction product of one or more pairs of conjugation handles and a complementary conjugation handle thereof. In some embodiments, the reaction product comprises a triazole, a hydrazone, pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, an alkene, or any combination thereof. In some embodiments, the reaction product comprises a triazole. The reaction product can be separated from the first point of attachment and the second point of attachment by any portion of the linker. In some embodiments, the reaction product is substantially in the center of the linker. In some embodiments, the reaction product is substantially closer to one point of attachment than the other is.

[0290] In some embodiments, the linker comprises a structure of Formula (X)1. I . L8. L7. L6. L5. L4. 1 . L2. L1. t wherein each of L1, L2, L3, L4, L5, L6, L7;L8, and L9is independently -O-, -NRL-, -(Ci-Ce alkylene)NRL-, -NRL(CI-C6alkylene)-, -N(RL)2+-, -(Ci-C6alkylene)N(RL)2+-, -N(RL)2+-(CI- C6alkylene)-, -OP(=O)(ORL)O-, -S-, -(Ci-C6alkylene)S-, -S(Ci-C6alkylene)-, -S(=O)-, - S(=O)2-, -C(=O)-, -(Ci-C6alkyl ene)C(=O)-, -C(=O) (Ci-C6alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NRL-, -C(=O)NRL(CI-C6alkylene)-, -(Ci-C6alkylene)C(=O)NRL-, - NRLC(=O)-, -(CI-C6alkylene)NRLC(=O)-, -NRLC(=O)(CI-C6alkylene)-, -OC(=O)NRL-, - NRLC(=O)O-, -NRLC(=O)NRL-, -NRLC(=S)NRL-, -CRL=N-, -N=CRL, -NRLS(=O)2-, - S(=O)2NRL-, -C(=O)NRLS(=O)2-, -S(=O)2NRLC(=O)-, substituted or unsubstituted Ci-Ce alkylene, substituted or unsubstituted Ci-Ce heteroalkylene, substituted or unsubstituted C2- Ce alkenylene, substituted or unsubstituted C2-Ce alkynylene, substituted or unsubstituted Ce- C2o arylene, substituted or unsubstituted C2-C2o heteroarylene, -(CH2-CH2-O)qa-, -(O-CH2- CH2)qb-, -(CH2-CH(CH3)-O)qc-, -(O- CH(CH3)-CH2)qd-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; each RLis independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-Ce alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C? heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qa, qb, qc and qd is independently an integer from 1-100,WSGR Docket No. 56146-747.601 wherein each is a point of attachment to the Fc domain or the cytokine (e.g., the IL-18 polypeptide).

[0291] In some embodiments, the linker consists of a plurality of structures of Formula (X) to form the linkage between the Fc domain and the cytokine (e.g., the IL-18 polypeptide) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more structures of Formula (X) appended from end to end, where only the terminal denote points of attachment to the Fc domain or the cytokine (e.g., IL-18 polypeptide)).

[0292] In some embodiments, the polymer comprises a linker comprising a structure of Formula(X’)HL'H wherein each L’ is independently -O-, -NRL-, -(Ci-Ce alkylene)NRL-, -NRL(Ci-Ce alkylene)-, - N(RL)2+-, -(CI-C6alkylene)N(RL)2+-, -N(RL)2+-(CI-C6alkylene)-, -OP(=O)(ORL)O-, -S-, - (Ci-C6alkylene)S-, -S(Ci-C6alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(Ci-C6alkylene)C(=O)- , -C(=O) (C1-C6 alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -C(=O)NRL-, -C(=O)NRL(CI- C6alkylene)-, -(Ci-C6alkylene)C(=O)NRL-, -NRLC(=O)-, -(Ci-C6alkylene)NRLC(=O)-, - NRLC(=O)(CI-C6alkylene)-, -OC(=O)NRL-, -NRLC(=O)O-, -NRLC(=O)NRL-, - NRLC(=S)NRL-, -CRL=N-, -N=CRL, -NRLS(=O)2-, -S(=O)2NRL-, -C(=O)NRLS(=O)2-, - S(=O)2NRLC(=O)-, substituted or unsubstituted Ci-Ce alkylene, substituted or unsubstituted Ci-Ce heteroalkylene, substituted or unsubstituted C2-C6 alkenylene, substituted or unsubstituted C2-C6 alkynylene, substituted or unsubstituted C6-C20 arylene, substituted or unsubstituted C2-C20 heteroarylene, -(CH2-CH2-O)qa-, -(O-CFh-CFDqb-, -(CH2-CH(CH3)- O)qc-, -(O- CH(CH3)-CH2)qd-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; (Ci-Ce alkylene); each RLis independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted CL-Cx cycloalkyl, substituted or unsubstituted C2-C7 heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qa, qb, qc and qd is independently an integer from 1-100, g is an integer from 1-100,WSGR Docket No. 56146-747.601 wherein each is a point of attachment to the cytokine (e.g., IL-18 polypeptide) or the Fc domain.

[0293] In some embodiments, the linker of Formula (X) or Formula (X’) comprises the structure:0is the attachment to a lysine residue of the Fc domain;L is a linking group; andpoint of attachment to a linking group which connects to point of attachment on the cytokine (e.g. IL-18 polypeptide), or a regioisomer thereof.WSGR Docket No. 56146-747.601wherein each n is independently an integer from 1-6 and each m is an integer from 1-30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1-24, from 1-18, from 1-12, or from 1-6.

[0295] In some embodiments, the linker of Formula (X) or of Formula (X’) comprises the structure:0is the first point of attachment to a lysine residue of the Fc domain of the immunocytokine composition;L” is a linking group; andpoint of attachment to a linking group which connects to the cytokine (e.g. theIL- 18 polypeptide), or a regioisomer thereof.WSGR Docket No. 56146-747.601independently an integer from 1-6 and each m is independently an integer from 1-30. In some embodiments, each m is independently 2 or 3. In some embodiments, each m is an integer from 1-24, from 1-18, from 1-12, or from 1-6.

[0297] In some embodiments, L or L” comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more subunits each independently, wherein each n is independently an integer from 1-30. In some embodiments, each n is independently an integer from 1-6. In some embodiments, L or L” comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the subunits.

[0298] In some embodiments, L or L” is a structure of Formula (X”)wherein each of Lla, L2a, L3a, L4a, L5a, is independently -O-, -NRLa-, -(Ci-Ce alkylene)NRLa-, - NRLa(Ci-C6alkylene)-, -N(RL)2+-, -(Ci-C6alkylene)N(RLa)2+(Ci-C6alkylene)-, -N(RL)2+-, - OP(=O)(ORLa)O-, -S-, -(Ci-C6alkylene)S-, -S(Ci-C6alkylene)-, -S(=O)-, -S(=O)2-, -C(=O)-, -(C1-C6 alkylene)C(=O)-, -C(=O)(Ci-C6alkylene)-, -C(=O)O-, -OC(=O)-, -OC(=O)O-, -WSGR Docket No. 56146-747.601C(=O)NRLa-, -C(=O)NRLa(Ci-C6alkylene)-, -(Ci-C6alkylene)C(=O)NRLa-, -NRLaC(=O)-, - (Ci-C6alkylene)NRLaC(=O)-, -NRLaC(=O)(Ci-C6alkylene)-, -OC(=O)NRLa-, -NRLaC(=O)O- , -NRLaC(=O)NRLa-, -NRLaC(=S)NRLa-, -CRLa=N-, -N=CRLa, -NRLaS(=O)2-, -S(=O)2NRLa-, - C(=O)NRLaS(=O)2-, -S(=O)2NRLaC(=O)-, substituted or unsubstituted Ci-Ce alkylene, substituted or unsubstituted Ci-Ce heteroalkylene, substituted or unsubstituted C2-Ce alkenylene, substituted or unsubstituted C2-Ce alkynylene, substituted or unsubstituted Ce-C2o arylene, substituted or unsubstituted C2-C2o heteroarylene, -(CH2-CH2-O)qe-, -(O-CH2-CH2)qf- , -(CH2-CH(CH3)-O)qg-, -(O- CH(CH3)-CH2)qh-, a reaction product of a conjugation handle and a complementary conjugation handle, or absent; (Ci-Ce alkylene) each RLais independently hydrogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 heteroalkyl, substituted or unsubstituted C2-Ce alkenyl, substituted or unsubstituted C2-C5 alkynyl, substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C2-C? heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and each of qe, qf, qg and qh is independently an integer from 1-100.

[0299] In some embodiments, L or L” comprises a linear chain of 2 to 10, 2 to 15, 2 to 20, 2 to 25, or 2 to 30 atoms. In some embodiments, the linear chain comprises one or more alkyl groups (e.g., lower alkyl (C1-C4)), one or more aromatic groups (e.g., phenyl), one or more amide groups, one or more ether groups, one or more ester groups, or any combination thereof.

[0300] In some embodiments, the linking group which connects to the point of attachment of the cytokine, (e.g., the IL- 18 polypeptide) comprises poly(ethylene glycol). In some embodiments, the linking group comprises about 2 to about 30 poly(ethylene glycol) units. In some embodiments, the linking group which connects to the point of attachment of the cytokine (e.g. the IL- 18 polypeptide) is a functionality attached to a cytokine provided herein which comprises an azide (e.g., the tri azole is the reaction product of the azide).

[0301] In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle independently comprises a triazole, a hydrazone, pyridazine, a sulfide, a disulfide, an amide, an ester, an ether, an oxime, or an alkene. In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle comprises a triazole. In some embodiments, each reaction product of a conjugation handle and a complementary conjugation handle comprise a structureWSGR Docket No. 56146-747.601regioisomer or derivative thereof.the carbonyl on the left side of the molecule is attached to an Fc domain lysine (e.g., K248) of the immunocytokine composition and the right side of the molecule is attached to a cysteine of the cytokine (e.g., C68 of the IL-18 polypeptide). In exemplified versions of the immunocytokines provided herein, the linker has the structurePeptide Linkers

[0303] In some embodiments, the Fc domain is linked to the other components of the immunocytokine composition (e.g., the binding domains or the cytokine, such as the IL- 18 polypeptide). In some embodiments, the Fc domain is linked to the cytokine (e.g., the IL- 18 polypeptide) as a fusion protein. In some embodiments, the Fc domain is linked to both binding domains as a fusion protein. In such instances, the linker (if present) comprises one or more peptide bonds between the Fc domain and the other group (e.g., the binding domain or the cytokine). In some embodiments, the linker between the Fc domain and the binding domain or the cytokine is a bond. In some embodiments, the linker between the Fc domain and the binding domain or cytokine is a linking peptide. Non-limiting examples of linking peptides include, but are not limited to (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25),WSGR Docket No. 56146-747.601(GGSG)n(SEQ ID NO: 26), or (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, a linking peptide can be GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30). In some embodiments, a binding domain or cytokine is fused to the C-terminal end of the Fc domain (optionally through a linking peptide). In some embodiments, the binding domain or cytokine is fused to the N-terminal end of the Fc domain (optionally through a linking peptide). In embodiments wherein multiple portions of the immunocytokine composition are fused to the Fc domain, each peptide linker is independently selected and can be the same or different.

[0304] In embodiments in which a binding domain or the cytokine is fused to the N-terminus of the Fc domain, the linking peptide desirably is or comprises a hinge region of an antibody. In some embodiments (e.g., those in which the binding domain is a Fab), the linking peptide is a hinge region. For example, when the Fc domain is derived from an IgGl, an IgGl hinge region or variant thereof can desirably be used to link the binding domain to the Fc domain (e.g., a hinge region having a sequence EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 26...

Claims

WSGR Docket No. 56146-747.601CLAIMSWHAT IS CLAIMED IS:

1. A multifunctional immunocytokine composition, comprising: a) a first binding domain targeting programmed cell death protein 1 (PD-1); b) a second binding domain targeting vascular endothelial growth factor A (VEGFA); and c) a cytokine, wherein each of the first binding domain, the second binding domain, and the cytokine are in covalent association.

2. The composition of claim 1, wherein the cytokine is selected from an interleukin, a TNF family cytokine, an interferon, a TGF-b family cytokine, and a chemokine.

3. The composition of claim 1 or 2, wherein the cytokine is an IL- 18 polypeptide.

4. The composition of claim 3, wherein the IL-18 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to the sequence set forth in SEQ ID NO: 701.

5. The composition of claim 3 or 4, wherein the IL- 18 polypeptide exhibits reduced binding to IL- 18 binding protein (IL-18BP) compared to wild type IL- 18 (WT IL- 18) (SEQ ID NO: 701).

6. The composition of any one of claims 3-5, wherein, wherein the IL- 18 polypeptide contains one or more amino acid substitutions that are located at residue positions selected from Y01, F02, E06, VI 1, C38, K53, D54, S55, T63, E69, K70, E85, C76, M86, T95, D98, and C127, wherein residue position numbering of the IL-18 polypeptides is based on SEQ ID NO: 701 as a reference sequence.

7. The composition of any one of claims 3-6, wherein the one or more amino acid substitutions in the IL-18 polypeptide are selected from Y01G, F02A, E06K, VI II, C38S, C38A, K53A, D54A, S55A, T63A, C68S, C68A, E69C, K70C, C76S, C76A, E85C, T95C, D98C, C127S, and C127A.

8. The composition of any one of claims 3-7, wherein the IL- 18 polypeptide contains an E06K amino acid substitutions.

9. The composition of any one of claims 3-8, wherein the IL-18 polypeptide contains aK53A amino acid substitution.

10. The composition of any one of claims 3-9, wherein the IL- 18 polypeptides contains a T63A amino acid substitution.

11. The composition of any one of claims 3-10, wherein the IL-18 polypeptide comprises a VI II amino acid substitution.WSGR Docket No. 56146-747.60112. The composition of any one of claims 3-11, wherein the IL-18 polypeptide comprises a substitution at one or more of residues C38, C68, C76, and C127.

13. The composition of any one of claims 3-12, wherein the IL-18 polypeptide comprises substitutions at residues C38, C76, and C127 or residues C38, C68, C76, and C127.

14. The composition of claim 13, wherein each substitution at residues C38, C68, C76, and C127 is independently selected from a serine or alanine substitution.

15. The composition of claim 13, wherein the IL-18 polypeptide comprises C38A, C76A, and C127A substitutions, or wherein the IL-18 polypeptide comprises C38A, C68A, C76A, and C127A substitutions.

16. The composition of claim 3 or 4, wherein the IL- 18 polypeptide comprises an amino acid sequence described in Table 12, or wherein the IL-18 polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 701-711.

17. The composition of any one of claims 3-16, wherein the IL- 18 polypeptide is an activatable IL-18 polypeptide which comprises an artificial polypeptide comprising a protease cleavage site attached to the N-terminus of the IL- 18 polypeptide.

18. The composition of claim 17, wherein cleavage at the protease cleavage site enhances an IL-18 related activity of the IL-18 polypeptide.

19. The composition of claim 17 or 18, wherein the artificial polypeptide is of the formula:BM-CS, wherein BM is a blocking moiety and CS is a peptide comprising the protease cleavage site.

20. The composition of claim 19, wherein the blocking moiety is an IL-18 propeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO: 731.

21. The composition of claim 20, wherein the IL- 18 propeptide comprises the sequence set forth in SEQ ID NO: 731, 733, 735, or 736.

22. The composition of any one of claims 17-21, wherein the protease cleavage site is cleaved by a protease which is found at higher concentrations and / or demonstrates higher proteolytic activity within the tumor microenvironment relative to non-tumor tissue.

23. The composition of any one of claims 17-22, wherein the protease is selected from: kallikrein, thrombin, chymase, carboxypeptidase A, an elastase, proteinase 3 (PR-3), granzyme M, urokinase plasminogen activator (uPA), a calpain, a matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), a fibroblast activation protein alpha (FAP), a matriptase, a plasminogen activator, a cathepsin, a caspase, a tryptase, and a tumor cell surface protease.WSGR Docket No. 56146-747.60124. The composition of any one of claims 17-23, wherein the protease cleavage site is comprised in a protease recognition sequence having at least 80%, 8%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a peptide sequence set forth in Table 4.

25. The composition of any one of claims 17-24, wherein the protease recognition sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 744, 745, 746, 747, 748, 749, 750, 752, 753, or 754.

26. The composition of any one of claims 17-25, wherein the cleavage of the artificial polypeptide at the protease cleavage site leaves no amino acid residues of the artificial polypeptide attached to the IL- 18 polypeptide, or wherein cleavage of the artificial polypeptide at the protease cleavage site leaves 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues of the artificial polypeptide attached to the IL-18 polypeptide.

27. The composition of any one of claims 17-26, wherein the artificial polypeptide comprises at least 2, 3, or 4 protease cleavage sites.

28. The composition of any one of claims 17-27, wherein the IL-18 polypeptide comprises substitutions of one or more amino acids at or near the N- or C-terminus of the IL-18 polypeptide which form part of a recognition sequence for the protease cleavage site.

29. The composition of claim 28, wherein the substitutions of the one or more amino acids at or near the N- or C-terminus of the IL-18 polypeptide comprises amino acid substitutions of residues 1, 2, and / or 3 of the IL-18 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence.

30. The composition of any one of claims 3-29, wherein the IL-18 polypeptide incorporated into the immunocytokine exhibits an ECso value for IFNy release in parental NK92 cells of at most about 10 nM, at most about 5 nM, at most about 2 nM, at most about 1 nM, at most about 0.5 nM, at most about 0.25 nM, or at most about 0.1 nM.

31. The composition of any one of claims 3-30, wherein the IL-18 polypeptide is in covalent association via a linker attached to a side chain of an amino acid residue of the IL- 18 polypeptide.

32. The composition of claim 31, wherein the linker is attached to residue 38, 68, 69, 70, 76, 78, 85, 86, 95, 98, 121, 127, or 144 of the IL-18 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence.

33. The composition of claim 32, wherein the linker is attached to residue 68 of the IL-18 polypeptide.

34. The composition of any one of claims 31-33, wherein the linker is attached to an Fc region of the composition, preferably wherein the linker is attached to the Fc region at a position of a K246 amino acid residue, a K248 amino acid residue, a K288 amino acid residue, aWSGR Docket No. 56146-747.601K290 amino acid residue, or a K317 amino acid residue of the Fc region (EU numbering) (e.g., conjugation using AJICAP™ technology).

35. The composition of claim 34, wherein the linker is attached to the K248 residue.

36. The composition of any one of claims 3-30, wherein the IL-18 polypeptide is in covalent association via a C-terminal fusion of the IL- 18 polypeptide to the portion of the composition to which it is attached, or wherein the IL- 18 polypeptide is in covalent association via an N-terminal fusion of the IL-18 polypeptide to the portion of the composition to which it is attached.

37. The composition of any one of claims 1-36, wherein the first binding domain targeting programmed cell death protein 1 (PD-1) is capable of disrupting the interaction of PD-1 with programmed cell death ligand 1 (PD-L1).

38. The composition of any one of claims 1-37, wherein the first binding domain is comprised in an antigen binding fragment derived from an antibody.

39. The composition of any one of claims 1-38, wherein the first binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3).

40. The composition of claim 39, wherein the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, abispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

41. The composition of claim 39 or 40, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 1 A, IB, or 1C.

42. The composition of any one of claims 39-41, wherein the VH comprise an amino acid sequence of a VH set forth in Table 1 A, IB, or 1C.

43. The composition of any one of claims 39-42, wherein the first binding domain is a VHH.

44. The composition of claim 43, wherein the first binding domain comprises: a) a VH CDR1 sequence of SEQ ID NO: 2; a VH CDR2 sequence of SEQ ID NO: 3; and a VH CDR3 sequence of SEQ ID NO: 4; b) a VH CDR1 sequence of SEQ ID NO: 6; a VH CDR2 sequence of SEQ ID NO: 7; and a VH CDR3 sequence of SEQ ID NO: 8; c) a VH CDR1 sequence of SEQ ID NO: 14; a VH CDR2 sequence of SEQ ID NO: 15; and a VH CDR3 sequence of CDR3 SEQ ID NO: 16;WSGR Docket No. 56146-747.601 d) a VH CDR1 sequence of SEQ ID NO: 18; a VH CDR2 sequence of SEQ ID NO: 19; and a VH CDR3 sequence of CDR 3 of SEQ ID NO: 20; e) a VH CDR1 sequence of SEQ ID NO: 288, a VH CDR2 sequence of SEQ ID NO: 289, and a VH CDR3 sequence of SEQ ID NO: 290, or f) a VH CDR1, VH CDR2, and VHCDR3 of a VHH provided in Table 1C.

45. The composition of claim 43 or 44, wherein the VHH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 1, 5, 13, 17, 287, or that of a VHH provided in Table 1C.

46. The composition of any one of claims 39-42, wherein the VH comprises a) a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82); b) a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88); or c) a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115).

47. The composition of any one of claims 39-42 or 46, wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 48, 50, or 76.

48. The composition of any one of claims 1-42 or 46-47, wherein the first binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

49. The composition of claim 48, wherein the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the first binding domain is comprised.

50. The composition of claim 48 or 49, wherein the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 1 A.

51. The composition of any one of claims 48-50, wherein the VL comprises:WSGR Docket No. 56146-747.601 a) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); b) a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); or c) a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).

52. The composition of any one of claims 48-51, wherein the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 49, 51, or 77.

53. The composition of any one of claims 1-42 or 46-52, wherein the first binding domain comprises: a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); or b) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91; or c) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118)54. The composition of any one of claims 1-42 or 46-53, wherein the first binding domain comprises: a) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 49;WSGR Docket No. 56146-747.601 b) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 50 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 51; c) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 76 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 77; d) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10; or e) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12.

55. The composition of any one of claims 1-42 or 46-54, wherein the first binding domain is an scFv.

56. The composition of any one of claims 1-42 or 46-54, wherein the first binding domain is a Fab.

57. The composition of any one of claims 1-56, wherein the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

58. The composition of any one of claims 1-57, wherein the second binding domain is comprised in an antigen binding fragment derived from an antibody.

59. The composition of any one of claims 1-58, wherein the second binding domain comprises a heavy chain variable domain (VH) comprising a heavy chain first complementary determining region (VH CDR1), a heavy chain second complementary determining region (VH CDR2), and a heavy chain third complementary determining region (VH CDR3).

60. The composition of claim 59, wherein the VH is comprised in a Fab, a Fab’, F(ab')2, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecific scFv, disulfide stabilized Fv (dsFv), a minibody, a diabody, abispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.WSGR Docket No. 56146-747.60161. The composition of claim 59 or 60, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 2 A, 2B, or 2C.

62. The composition of any one of claims 59-61, wherein the VH comprise an amino acid sequence of a VH set forth in Table 2A, 2B, or 2C.

63. The composition of any one of claims 59-62, wherein the VH comprises a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125).

64. The composition of any one of claims 59-63, wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 122.

65. The composition of any one of claims 1-64, wherein the second binding domain comprises a light chain variable domain (VL) comprising a light chain first complementary determining region (VL CDR1), a light chain second complementary determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

66. The composition of claim 65, wherein the VL is comprised in the Fab, Fab’, F(ab')2, bispecific F(ab')2, variable fragment (Fv), single chain variable fragment (scFv), bispecific scFv, disulfide stabilized Fv (dsFv), minibody, diabody, bispecific diabody, triabody, tetrabody, maxibody, Fab-Fc, scFv-Fc, or bispecific antibody in which the VH of the second binding domain is comprised.

67. The composition of claim 65 or 66, wherein the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2A.

68. The composition of any one of claims 65-67, wherein the VL comprises a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130).

69. The composition of any one of claims 65-68, wherein the VL comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 127.

70. The composition of any one of claims 1-69, wherein the second binding domain comprises a VH having a VH CDR1 having a sequence GYTFTNYGMN (SEQ ID NO: 123), a VH CDR2 having a sequence WINTYTGEPTYAADFK (SEQ ID NO: 124), and a VH CDR3 having a sequence YPHYYGSSHWYFDV (SEQ ID NO: 125), and a VL having a VL CDR1 having a sequence SASQDISNYLN (SEQ ID NO: 128), a VL CDR2 having aWSGR Docket No. 56146-747.601 sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130).

71. The composition of any one of claims 1-70, wherein the second binding domain comprises a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 127.

72. The composition of any one of claims 1-71, wherein the second binding domain is an scFv.

73. The composition of any one of claims 1-72, wherein the second binding domain is a Fab.

74. The composition of any one of claims 1-58, wherein the second binding domain is a single domain antibody.

75. The composition of claim 74, wherein the second binding domain is a single domain heavy chain antibody (VHH).

76. The composition of claim 74 or 75, wherein the second binding domain comprises: a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204); b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208); c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFDY (SEQ ID NO: 212); d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); e) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (S EQ ID NO; 219 ) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220); or f) a VH CDR I, VH CDR2, and VH CDR3 of a VHH provided in Table 2C.

77. The composition of any one of claims 74-76, wherein the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, or 280-284, or the a VH sequence provided in Table 2C.WSGR Docket No. 56146-747.60178. The composition of claim 74, wherein the second binding domain comprises a light chain single domain antibody.

79. The composition of claim 74 or 78, wherein the second binding domain comprises a VL CDR1 having the sequence RASQWIGPELS (SEQ ID NO: 223), a VL CDR2 having the sequence HTSILQS (SEQ ID NO: 224), and a VL CDR3 having the sequence QQYMFQPRT (SEQ ID NO: 225).

80. The composition of any one of claims 74, 78, or 79 wherein the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 222.

81. The composition of any one of claims 1-57, wherein the second binding domain is an anti- VEGFA anticalin.

82. The composition of any one of claims 1-57 or 81, wherein the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95&, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 226.

83. The composition of any one of claims 1-82, wherein the composition comprises an Fc domain comprising first CH2 and CH3 domains on a first polypeptide chain and second CH2 and CH3 domains on a second polypeptide chain.

84. The composition of claim 83, wherein the Fc domain is derived from an IgG.

85. The composition of claim 84, wherein the Fc domain is derived from an IgGl or IgG4.

86. The composition of any one of claims 83-85, wherein the composition comprises a structure of the formula:C X-Y-Z X'-Y-Z' i c wherein:Y is the first CH2 and CH3 domains;Y’ is the second CH2 and CH3 domains;X and X’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, the cytokine, a copy of the cytokine, or absent;Z and Z’ are each independently the first binding domain, the second binding domain, a copy of the first binding domain, a copy of the second binding domain, the cytokine, a copy of the cytokine, a third binding domain targeting PD-1, or a fourth binding domain targeting VEGFA, or absent.WSGR Docket No. 56146-747.601C and C’ are each independently the cytokine, a copy of the cytokine, or absent, wherein C and C’, if present, are attached to a side chain of a residue of the Fc domain via a linker; wherein X, Y, and Z and X’, Y’ and Z’ are depicted in an N-terminal to C-terminal direction; and wherein each of X, X’, Z, and Z’ are independently and optionally connected to Y or Y’ via a peptide linker.

87. The composition of claim 86, wherein X is the first binding domain; X’ is a copy of the first binding domain, the cytokine, or absent; one of Z or Z’ is the second binding domain and the other is absent or a copy of the second binding domain; and one of C or C’ is the cytokine and the other is absent, or both C and C’ are absent.

88. The composition of claim 87, wherein X’ is the cytokine and C and C’ are both absent.

89. The composition of claim 87, wherein X’ is the copy of the first binding domain and one of C or C’ is the cytokine.

90. The composition of any one of claims 87-89, wherein:Z is the second binding domain and Z’ is a copy of the second binding domain;Z is the second binding domain and Z’ is absent; orZ is absent and Z’ is the second binding domain.

91. The composition of claim 86, wherein X is the second binding domain, X’ is a copy of the second binding domain, the cytokine, or absent; one of Z or Z’ is the first binding domain and the other is absent or a copy of the first binding domain; and one of C or C’ is the cytokine and the other is absent, or both C and C’ are absent.

92. The composition of claim 91, wherein X’ is the cytokine and C and C’ are both absent.

93. The composition of claim 91, wherein X’ is a copy of the second binding domain one of C or C’ is the cytokine.

94. The composition of any one of claims 86-89, wherein:Z is the first binding domain and Z’ is a copy of the first binding domain;Z is the first binding domain and Z’ is absent; orZ is absent and Z’ is the first binding domain.

95. The composition of claim 86, wherein X is the first binding domain, X’ is the second binding domain, one of C or C’ is the cytokine and the other is absent, Z is absent or the third binding domain, and Z’ is absent or the fourth binding domain.

96. The composition of claim 95, wherein:Z is the third binding domain and Z’ is absent;Z is absent and Z’ is the fourth binding domain; orWSGR Docket No. 56146-747.601 both Z and Z’ are absent.

97. The composition of any one of claims 86 or 94-96, wherein if the third binding domain is present, the first binding domain comprises a Fab and the third binding domain comprises an scFv, and wherein the Fab and the scFv comprise the same VH and VL.

98. The composition of any one of claims 86 or 94-96, wherein if the fourth binding domain is present, the second binding domain comprises a Fab and the fourth binding domain comprises an scFv, and wherein the Fab and the scFv comprise the same VH and VL.

99. The composition of any one of claims 86-98, wherein X is one of the first or second binding domains and is a Fab, VHH, or scFv.

100. The composition of claim 99, wherein X is one of the first or second binding domains and is a Fab.

101. The composition of claim 99 or 100, wherein X’ is a copy of X.

102. The composition of any one of claims 99-101, wherein one Z or Z’ is one of the first or second binding domains and is an scFv or a VHH, wherein if X is the first binding domain then Z or Z’ is the second binding domain and if X is the second binding domain then Z or Z’ is the first binding domain.

103. The composition of claim 86, wherein:X is a Fab and the first binding domain, X’ is a copy of the first binding domain, Z is an scFv or VHH and is the second binding domain, Z’ is a copy of the second binding domain, C is the cytokine, and C’ is absent; orX is a Fab and the second binding domain, X’ is a copy of the second binding domain, Z is an scFv or VHH and is the first binding domain, Z’ is a copy of the first binding domain, C is the cytokine, and C’ is absent; orX is a Fab and is the first binding domain, X’ is a Fab and is the second binding domain, Z and Z’ are absent, and C or C’ is the cytokine and the other is absent;X is a Fab and is the first binding domain, X’ is a Fab and is the second binding domain; Z is an scFv or VHH and is the third binding domain, Z’ is absent, and C or C’ is the cytokine and the other is absent; orX is a Fab and is the second binding domain, X’ is a Fab and is the first binding domain, Z is an scFv or VHH and is the fourth binding domain, Z’ is absent, and C or C’ is the cytokine and the other is absent; orX is a Fab and is the first binding domain, X’ is the cytokine, Z is an scFv or VHH and is the second binding domain, Z’ is a copy of the second binding domain, and C and C’ are both absent; orWSGR Docket No. 56146-747.601X is a Fab and is the second binding domain, X’ is the cytokine, Z is an scFv or VHH and is the first binding domain, Z’ is a copy of the first binding domain, and C and C’ are both absent; orX is a Fab and is the first binding domain, X’ is an scFv and is the second binding domain; Z and Z’ are both absent, and C or C’ is the cytokine and the other is absent; orX is a Fab and is the second binding domain, X’ is an scFv and is the first binding domain, Z and Z’ are both absent, and C or C’ is the cytokine and the other is absent.

104. The composition of any one of claims 1-103, wherein the composition comprises only one cytokine.

105. The composition of any one of claims 83-103, wherein the Fc domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence set forth in any one of SEQ ID NOs: 229-234 or 236-241.

106. The composition of any one of claims 3-82, wherein the first binding domain comprises a Fab, and wherein the composition comprises: a) a first polypeptide chain comprising the VL of the first binding domain; b) a second polypeptide chain comprising the VH of the first binding domain; and c) a third polypeptide chain comprising the IL-2 polypeptide.

107. The composition of claim 106, wherein the first binding domain comprises: a) a VH having a VH CDR1 sequence of NYYMY (SEQ ID NO: 80), a VH CDR2 sequence of GINPSNGGTNFNEKFKN (SEQ ID NO: 81), and a VH CDR3 sequence of RDYRFDMGFDY (SEQ ID NO: 82), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLES (SEQ ID NO: 84), and a VL CDR3 sequence of QHSRDLPLT (SEQ ID NO: 85); or b) a VH having a VH CDR1 sequence of NSGMH (SEQ ID NO: 86), a VH CDR2 sequence of VIWYDGSKRYYADSVKG (SEQ ID NO: 87), and a VH CDR3 sequence of NDDY (SEQ ID NO: 88), and a VL having a VL CDR1 sequence of RASQSVSSYLA (SEQ ID NO: 89), a VL CDR2 sequence of DASNRAT (SEQ ID NO: 90), and a VL CDR3 sequence of QQSSNWPRT (SEQ ID NO: 91); or c) a VH having a VH CDR1 sequence of GYTFTSYYMY (SEQ ID NO: 113), a VH CDR2 sequence of GVNPSNGGTNFNEKFKS (SEQ ID NO: 114), and a VH CDR3 sequence of RDYRYDMGFDY (SEQ ID NO: 115), and a VL having a VL CDR1 sequence of RASKGVSTSGYSYLH (SEQ ID NO: 83), a VL CDR2 sequence of LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).WSGR Docket No. 56146-747.601108. The composition of claim 106 or 107, wherein the first binding domain comprises: a) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 49; b) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 50 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 51; or c) a VH comprising an amino acid sequence amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 76 and a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 77.

109. The composition of any one of claims 106-108, wherein the first polypeptide chain comprises, in N- to C-terminal direction, the VL and light chain constant region.

110. The composition of claim 109, wherein the light chain constant region comprises an amino acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 276.

111. The composition of any one of claims 106-110, wherein the first polypeptide chain comprises the sequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLA SYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQE SVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

112. The composition of any one of claims 106-111, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab of the first binding domain and an antibody constant region.

113. The composition of claim 112, wherein the antibody constant region is an IgGl or IgG4 constant region.

114. The composition of claim 112 or 113, wherein the antibody constant region comprises, in an N-terminal to C-terminal direction, a CHI domain, a hinge region, a CH2 domain, and a CH3 domain.

115. The composition of claim 114, whereinWSGR Docket No. 56146-747.601 a) the CHI domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 272-275; b) the hinge region comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 265-271; and / or c) the CH2 and CH3 domains together comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 229-234.

116. The composition of any one of claims 112-115, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab, the antibody constant region, an optional peptide linker, and the second binding domain.

117. The composition of claim 116, wherein the optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

118. The composition of claim 116 or 117, wherein the second binding domain is a VHH.

119. The composition of claim 118, wherein the VHH comprises a two proline peptide on its C-terminus.

120. The composition of claim 118 or 119, wherein the VHH comprises a) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVRG (SEQ ID NO: 203), and a VH CDR3 sequence of DPRKLDY (SEQ ID NO: 204); b) a VH CDR1 sequence of LYDMM (SEQ ID NO: 206), a VH CDR2 sequence of FIGGDGLNTYYADSVKG (SEQ ID NO: 207), and a VH CDR3 sequence of AGTQFDY (SEQ ID NO: 208); c) a VH CDR1 sequence of WYPMW (SEQ ID NO: 210), a VH CDR2 sequence of LIEGQGDRTYYADSVKG (SEQ ID NO: 211), and a VH CDR3 sequence of AGDRTAGSRGNSFDY (SEQ ID NO: 212); d) a VH CDR1 sequence of AYPMM (SEQ ID NO: 202), a VH CDR2 sequence of EISPSGSYTYYADSVKG (SEQ ID NO: 215), and a VH CDR3 sequence of DPRKFDY (SEQ ID NO: 216); orWSGR Docket No. 56146-747.601 e) a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

121. The composition of any one of claims 118-121, wherein the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 200, 201, 205, 209, 213, 217, 221, or 280-284.

122. The composition of any one of claims 118-121, wherein the VHH comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

123. The composition of claim 122, wherein the second binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:200, 217, or 221.

124. The composition of any one of claims 116-123, wherein the second polypeptide chain further comprises an additional binding domain targeting VEGFA.

125. The composition of any one of claims 116-124, wherein the additional binding domain targeting VEGFA is a VHH.

126. The composition of claim 124 or 125, wherein both the second binding domain and the additional binding domain targeting VEGFA are both VHHs comprising an identical amino acid sequence, or wherein the VHH positioned C-terminal to the other VHH comprises an additional two proline peptide on its C-terminus as compared to the other VHH, optionally wherein the VHH comprises a two proline peptide on its C-terminus.

127. The composition of claim 125 or 126, wherein the additional binding domain targeting VEGFA comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SILAYGSSRLRLADTYEY (SEQ ID NO: 220).

128. The composition of any one of claims 125-127, wherein the additional binding domain targeting VEGFA an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200, 217, or 221.

129. The composition of any one of claims 124-128, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab, an antibodyWSGR Docket No. 56146-747.601 constant region, an optional peptide linker, the second binding domain, a second optional peptide linker, and the additional binding domain targeting VEGFA.

130. The composition of claim 129, wherein the second optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

131. The composition of any one of claims 106-130, wherein the second polypeptide chain comprises the sequence set forth in any one of SEQ ID NOs: 163, 164, 169, 171, or 172.

132. The composition of any one of claims 106-131, wherein the third polypeptide chain comprises, in an N-terminal to C-terminal direction, the IL-18 polypeptide, an optional peptide linker, and an antibody constant region.

133. The composition of claim 132, wherein the optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

134. The composition of any one of claims 132 or 133, wherein the third polypeptide chain comprises, in an N-terminal to C-terminal direction, the IL-2 polypeptide, the optional peptide linker, an antibody constant region, a second optional peptide linker, and the second binding domain or an additional binding domain targeting VEGFA, wherein the additional binding domain targeting VEGFA is present if the second binding is present on the second polypeptide chain or if the second binding domain is also present on the third polypeptide chain.

135. The composition of claim 134, wherein the second optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

136. The composition of claim 134 or 135, wherein the second binding domain or the additional binding domain targeting VEGFA is a VHH.WSGR Docket No. 56146-747.601137. The composition of claim 136, wherein the VHH comprises a two proline peptide on its C-terminus.

138. The composition of claim 136 or 137, wherein the second binding domain or the additional binding domain targeting VEGFA comprises a VH CDR1 having the sequence SYSMG (SEQ ID NO: 218), a VH CDR2 having the sequence AISKGGYKYDAVSLEG (SEQ ID NO: 219) or the sequence of SEQ ID NO: 279, and a VH CDR3 having the sequence SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

139. The composition of claim 138, wherein the second binding domain or the additional binding domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 200, 217 or 221.

140. The composition of any one of claims 132-139, wherein the third polypeptide comprises the second binding domain and the additional binding domain targeting VEGFA separated by an optional peptide linker.

141. The composition of claim 140, wherein the optional peptide linker is present and has a sequence (GS)n(SEQ ID NO: 23), (GGS)n(SEQ ID NO: 24), (GGGS)n(SEQ ID NO: 25), (GGSG)n(SEQ ID NO: 26), (GGSGG)n(SEQ ID NO: 27), (GGGGS)n(SEQ ID NO: 28), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a sequence GGGGS (SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

142. The composition of claim 140 or 141, wherein both the second binding domain and the additional binding domain targeting VEGFA are both VHHs comprising an identical amino acid sequence, or wherein the VHH positioned C-terminal to the other VHH comprises an additional two proline peptide on its C-terminus as compared to the other VHH.

143. The composition of any one of claims 132-142, wherein the antibody constant region is an IgGl or IgG4 constant region, or a portion thereof.

144. The composition of any one of claims 132-143, wherein the antibody constant region comprises, in an N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain.

145. The composition of claim 143 or 144, wherein the antibody constant region comprises, in an N-terminal to C-terminal direction, a hinge region portion, a CH2 domain, and a CH3 domain.

146. The composition of claim 145, whereinWSGR Docket No. 56146-747.601 a) the hinge region portion comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs 265-271; and / or b) the CH2 and CH3 domains together comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 229-234.

147. The composition of any one of claims 132-146, wherein the third polypeptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 722 or 723.

148. The composition of claim 106, wherein: the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 47; the second polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 163, 164, 169, 171, or 172; and the third polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 722 or 723.

149. The composition of claim 106, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, the amino acid sequences set forth in:SEQ ID NOs: 47, 163, and 722;SEQ ID NOs: 47, 163, and 723;SEQ ID NOs: 47, 164, and 722;SEQ ID NOs: 47, 164, and 723;SEQ ID NOs: 47, 169, and 722;SEQ ID NOs: 47, 169, and 723;SEQ ID NOs: 47, 171, and 722;SEQ ID NOs: 47, 171, and 723;SEQ ID NOs: 47, 172, and 722; orSEQ ID NOs: 47, 172, and 723.

150. The composition of claim 106, wherein the first polypeptide chain, the second polypeptide chain, and the third polypeptide chain comprise, respectively, the amino acid sequences set forth in SEQ ID NOs: 47, 164, and 722.

151. An immunocytokine composition comprising: a binding domain targeting vascular endothelial growth factor A (VEGFA); and an IL- 18 polypeptide.WSGR Docket No. 56146-747.601152. The composition of claim 151, wherein the IL-18 polypeptide is any of the IL-18 polypeptides described herein.

153. The composition of claim 151 or 152, wherein the binding domain targeting VEGFA is any of those described herein.

154. The composition of any one of claims 151-153, wherein the binding domain targeting VEGFA is an antibody or an antigen binding fragment thereof.

155. The composition of any one of claims 151-154, wherein the binding domain targeting VEGFA is a monoclonal antibody.

156. The composition of any one of claims 151-155, further comprising a binding domain targeting PD-1.

157. The composition of claim 156, wherein the binding domain targeting PD-1 is any one of the binding domains targeting PD-1 described herein.

158. One or more polynucleotides encoding the composition of any one of the preceding claims, or a portion thereof.

159. A host cell comprising the composition of claims 1-157 or the one or more polynucleotides of claim 158.

160. A pharmaceutical composition comprising the composition of any one of claims 1- 157, and a pharmaceutically acceptable carrier or excipient.

161. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1-157 or the pharmaceutical composition of claim 160.

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