Trispecific compositions comprising il-2, VEGF binding domains, and PD-1 binding domains

Abstract

The present disclosure relates anti-PD-1 / anti-VEGF immunocytokines, particularly with IL-2. Also described herein are bispecific anti-PD-1 and anti-VEGF constructs. IL-2 polypeptides are also provided herein. Also provided herein are pharmaceutical compositions and methods of treatment using the aforementioned compositions.

Description

Docket No. 56146-746.601TRISPECIFIC COMPOSITIONS COMPRISING IL-2, VEGF BINDING DOMAINS, AND PD-1 BINDING DOMAINSCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 708,077, filed October 16, 2024, and U.S. Provisional Application No. 63 / 739,330, filed December 27, 2024, which applications are incorporated by reference in their entirety.BRIEF SUMMARY

[0002] Described herein are multifunctional immunocytokine compositions which comprise a PD-1 binding domain, a VEGFA binding domain, and a cytokine. In some embodiments, the cytokine is an IL-2 polypeptide. Such compositions are useful in the treatment of diseases and disorders such as cancer.

[0003] In some embodiments of a multifunctional immunocytokine composition, the VEGFA binding domain and PD-1 binding domain and / or IL-2 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-2 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-2 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-2 to a subject with increased therapeutic index by optimizing the activity in tumor microenvironments where VEGFA is present.

[0004] In some embodiments, a multifunctional immunocytokine of the instant disclosure utilizes an IL-2 polypeptide with reduced activity compared to wild type IL-2 (SEQ ID NO: 701, human IL-2). In some embodiments, use of such an IL-2 polypeptide allows for reduced off-target effects as the IL-2 can be sufficiently active due to the targeting nature of the other components of the immunocytokine and / or the multimerization effects described supra, yet will be less active outside of target tissue, thereby enhancing therapeutic index.

[0005] In some embodiments, an IL-2 polypeptide of the instant disclosure exhibits reduced binding to heparin compared to other IL-2 polypeptides or to wild type IL-2. In someDocket No. 56146-746.601 embodiments, the reduced binding to heparin can impart favorable characteristics to the immunocytokine composition, such as enhanced PK, half-life, bioavailability, and biodistribution due to the prevention of accumulation of the immunocytokine composition in heparin rich tissues.

[0006] Also described herein are novel anti-VEGFA and PD-1 binding domains with certain advantages over those otherwise known. Such binding domains in some instances have optimal properties for inclusion in an immunocytokine composition of the instant disclosure.

[0007] Further provided herein are methods of treating cancer and other disease with the aforementioned compositions, as well as pharmaceutical compositions comprising the same.

[0008] In an aspect, the present disclosure provides a 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.

[0009] 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.

[0010] In some embodiments, the cytokine is an IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to wild type IL-2 (SEQ ID NO: 701). In some embodiments, the IL-2 polypeptide has reduced affinity for the IL-2 receptor beta subunit compared to the IL-2 of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide exhibits a reduced ability to signal through the IL-2 receptor beta / gamma complex compared to the IL- 2 of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide retains the ability to bind to the IL-2 receptor alpha subunit and exhibits a diminished ability to bind to the IL-2 receptor beta or gamma subunits relative to SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide has reduced affinity for heparin compared to the IL-2 of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises a modified B’C’ loop region, wherein the modified B’C’ loop region comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide and insertion of an exogenousDocket No. 56146-746.601 peptide into the B’C’ loop region, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the inserted peptide comprises the sequence GDGSIN (SE ID NO: 700). In some embodiments, the inserted peptide consists of the sequence GDGSIN. In some embodiments, the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acids 73 and 84 of the IL-2 polypeptide. In some embodiments, the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of each of amino acids 74-83 of the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-73 of SEQ ID NO: 701 and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 501. In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 783. In some embodiments, the IL-2 polypeptide comprises a K76A or R81S substitution, or both. In some embodiments, the IL-2 polypeptide comprises polypeptide comprises an amino acid substitution at residue N88. In some embodiments, the IL-2 polypeptide comprises an N88D substitution. In some embodiments, the IL-2 polypeptide comprises a substitution at any one of residues L12, E15, L19, T123, Q126, or 1129. In some embodiments, the IL-2 polypeptide comprises one or more substitutions selected from L12A, L12Y, E15D, E15S, L19A, L19D, T123A, Q126T, I129A, and I129K. In some embodiments, the IL-2 polypeptide comprises any one of the following sets of substitutions: Q126T; I129K; I129A, E15S, T123A; E15D; L12A, L19A, E15S; L12Y, L19D; L12A, L19A; or L19D. In some embodiments, the IL-2 polypeptide comprises an E15D or an L19D substitution. In some embodiments, the IL-2 polypeptide comprises a T3A substitution. In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the composition comprises the sequence set forth in any one of SEQ ID NOs: 702-783. In some embodiments, the IL-2 polypeptide is in covalent association via a fusion of the IL-2 polypeptide to the portion of the composition to which it is attached. In some embodiments, the IL-2 polypeptide is fused via its C-terminus.

[0011] 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 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 complementaryDocket No. 56146-746.601 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 1A , IB, or 1C.

[0012] 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 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; 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.

[0013] 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 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 RDYRYDMGFD Y (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.

[0014] 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 thirdDocket No. 56146-746.601 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 or IB. 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 LASYLE (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.

[0015] 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 RDYRFDMGFD Y (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). 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%,Docket No. 56146-746.60185%, 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.

[0016] In some embodiments, the first binding domain is an scFv. In some embodiments, the first binding domain is a Fab.

[0017] 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.

[0018] 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 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 2A or 2B. In some embodiments, the VH comprises 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 isDocket No. 56146-746.601 comprised. In some embodiments, the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2A or 2B. 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.

[0019] In some embodiments, the second binding domain is an scFv. In some embodiments, the second binding domain is a Fab.

[0020] 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 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 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%,Docket No. 56146-746.60198%, 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 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.

[0021] 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.

[0022] In some embodiments, the composition comprises multiple copies of the first binding domain, the second binding domain, or both. In some embodiments, the composition comprises multiple copies of the second binding domain. In some embodiments, the composition comprises two copies of the second binding domain. In some embodiments, one of the copies of the second binding domain contains an extension of one or more amino acids on the second binding domain relative to the other copy. In some embodiments, the multiple copies of the second binding domain are present on the same polypeptide chain. In some embodiments, the multiple copies of the second binding domain are present on different polypeptide chains.

[0023] 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. In some embodiments, theCX-Y-ZX'-Y-Z' i 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, a masking polypeptide for 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, a masking polypeptide for the cytokine, or absent. C and C’ are eachDocket No. 56146-746.601 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.

[0024] 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, the cytokine, 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, X’ is the copy of the first binding domain, one of Z or Z’ is the second binding domain and the other is the cytokine, and both C and C’ are absent. 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, the cytokine, 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, X’ is the copy of the second binding domain, one of Z or Z’ is the second binding domain and the other is the cytokine, and both C and C’ are absent. 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, X is the first binding domain, X’ is the second binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and both C and C’ are absent. In some embodiments, the masking polypeptide for the cytokine is attached to the Y or Y’ to which it is connected via a cleavable peptide linker. 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 aDocket No. 56146-746.601Fab and the fourth binding domain comprises 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.

[0025] 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; or X is a Fab and is the first binding domain, X’ is Fab or ScFv and is the second binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and C and C’ are both absent; or X is a Fab and is the second binding domain, X’ is a Fab or scFv and is the first binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and C and C’ are both absent.

[0026] 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 bindingDocket No. 56146-746.601 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.

[0027] 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 RDYRFDMGFD Y (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). 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 sequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYL ESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPDocket No. 56146-746.601SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 47).

[0028] In 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) 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. 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 SRAYGSSRLRLADTYEY (SEQ ID NO: 220). In some embodiments, the second bindingDocket No. 56146-746.601 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 binding 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)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, or 171.

[0029] In some embodiments, the third polypeptide chain comprises, in an N-terminal to C- terminal direction, the IL-2 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 IDDocket No. 56146-746.601NO: 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, 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, 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, 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,Docket No. 56146-746.601 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: 802, 803, 806, or 807. 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, or 171; and the third polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 802, 803, 806, or 807. 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, 163, and 802;SEQ ID NOs: 47, 163, and 803; SEQ ID NOs: 47, 164, and 806;SEQ ID NOs: 47, 164, and 807; SEQ ID NOs: 47, 169, and 802;SEQ ID NOs: 47, 169, and 803; SEQ ID NOs: 47, 172, and 802;SEQ ID NOs: 47, 172, and 803; SEQ ID NOs: 47, 163, and 806;SEQ ID NOs: 47, 163, and 807; SEQ ID NOs: 47, 169, and 806;SEQ ID NOs: 47, 169, and 807; SEQ ID NOs: 47, 171, and 809; orSEQ ID NOs: 47, 171, and 810.

[0030] In some embodiments, the second binding domain is a Fab having a VH and a VL, 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.

[0031] In some embodiments, second binding domain Fab comprises a VH having a VH CD1, VH CDR2, and VH CDR3 and a VL having a VL CDR1, CDR2, and CDR3 of any one of the anti-VEGFA antibodies in Table 2A, optionally wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding VL. In some embodiments, the VH CDR1, VH CDR2, and VH CDR3 are SEQ ID NOs: 123, 124, and 125, respectively and the VL CDR1, VL CDR2, and VL CDR3 are SEQ ID NOs: 128, 129, and 130, respectively. In some embodiments, the VH and VL each have a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 122 and 126, respectively. In some embodiments, the first polypeptide chainDocket No. 56146-746.601 comprises, in N- to C-terminal direction, the VL and a 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, 277, or 278.

[0032] In some embodiments, the second polypeptide chain comprises, in an N-terminal to C- terminal direction, the VH of the Fab of the second binding domain and an antibody constant region. In some embodiments, the antibody constant region is an IgGl or an IgG4. 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, 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, 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 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 first binding domain specific for PD-1. In some embodiments, the optional peptide linker is present and comprises a sequence of any one of SEQ ID NOs: 21-30. In some embodiments, the first binding domain is a VHH. In some embodiments, the VHH comprises a VH CDR1, VH CDR2, and VH CDR3 of any one of the anti-PD-1 VHH in Table IB or Table 1C. In some embodiments, the anti-PD-1 binding domain comprises the CDRS of VHH47, VHH62, VHH70, VHH76, VHH84, or VHH178. In some embodiments, the first 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 VHH47, VHH62, VHH70, VHH76, VHH84, or VHH178. In some embodiments, the first binding domain is VHH47. In some embodiments, the second polypeptide chain further comprises an additional binding domain targeting PD-1. In some embodiments, the additional binding domain VHH comprises an identical amino acid sequence compared to the first binding domain. In some embodiments, the first binding domain and the additional binding domain are separated by a peptide linker, optionally wherein the peptide linker comprises a sequence of any one of SEQ ID NOs: 22-30.

[0033] In some embodiments, the third polypeptide chain comprises, in an N-terminal to C- terminal direction, a VH of a second Fab specific for VEGFA, an antibody constant region, an optional peptide linker, and the IL-2 polypeptide. In some embodiments, the second Fab is theDocket No. 56146-746.601 same as the Fab of the second binding domain. In some embodiments, the antibody constant region of the third polypeptide is an IgGl or an IgG4.. In some embodiments, the antibody constant region of the third polypeptide 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, 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, 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 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 optional peptide linker of the third polypeptide is present and comprises a sequence of any one of SEQ ID NOs: 21-30.

[0034] In some embodiments, the IL-2 polypeptide comprises the sequence set forth in SEQ ID NO: 751, 753, 754, or 758.

[0035] In some embodiments, the composition comprises only one cytokine.

[0036] In an aspect, the present disclosure provides one or more polynucleotides encoding the composition of any one of the embodiments disclosed herein, or a portion thereof.

[0037] In an aspect, the present disclosure provides a host cell comprising the composition of any one of the embodiments disclosed herein or the one or more polynucleotides of any of the embodiments disclosed herein.

[0038] In an aspect, the present disclosure provides a pharmaceutical composition comprising the composition of any one of the embodiments disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0039] In an aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject at therapeutically effective amount of the composition of any one of the embodiments disclosed herein or the pharmaceutical composition of any one of the embodiments disclosed herein.

[0040] In an aspect, the present disclosure provides an IL-2 polypeptide comprising a modified B’C’ loop region, wherein the modified B’C’ loop region comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence, and an insertion of a peptide comprising the sequence GDGSIN into the deleted portion of the B’C’ loop region. In some embodiments, the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acidsDocket No. 56146-746.60173 and 84 of the IL-2 polypeptide. In some embodiments, the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of each of amino acids 74-83 of the IL-2 polypeptide. In some embodiments, the inserted peptide consists of the sequence GDGSIN. In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-73 of SEQ ID NO: 701 and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 783. An IL-2 polypeptide comprising the amino acid substitutions K76A and R81S, wherein residue position numbering is based on SEQ ID NO: 1 as a reference sequence. In some embodiments, the IL-2 polypeptide exhibits reduced binding to heparin compared to the IL-2 polypeptide of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide exhibits reduced binding to the IL-2 receptor beta subunit compared to the IL-2 of SEQ ID NO: 1. In some embodiments, the IL-2 polypeptide comprises an amino acid substitution at residue N88. In some embodiments, the IL-2 polypeptide comprises an N88D substitution. In some embodiments, the IL-2 polypeptide exhibits reduced binding to the IL-2 receptor gamma subunit. In some embodiments, the IL-2 polypeptide comprises a substitution at any one of residues L12, E15, L19, T123, Q126, or 1129. In some embodiments, the IL-2 polypeptide comprises one or more substitutions selected from L12A, L12Y, E15D, E15S, L19A, L19D, T123A, Q126T, or 1129 A, I129K. In some embodiments, the IL-2 polypeptide comprises any one of the following sets of substitutions: Q126T; I129K; I129A, E15S, T123A; E15D; L12A, L19A, E15S; L12Y, L19D; L12A, L19A; or L19D. In some embodiments, the IL-2 polypeptide comprises an E15D or an L19D substitution. In some embodiments, the IL-2 polypeptide comprises a T3A substitution. In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the IL-2 polypeptide binds to the IL-2 receptor alpha subunit. In some embodiments, the IL-2 polypeptide comprises the sequence set forth in any one of SEQ ID NOs: 703-774.

[0041] In an aspect, the present disclosure provides a fusion polypeptide comprising the IL-2 polypeptide of any one of the embodiments disclosed herein and an additional polypeptide. In some embodiments, the IL-2 polypeptide is fused to the additional polypeptide at its C- terminus. In some embodiments, the additional polypeptide comprises an Fc domain. In some embodiments, the IL-2 polypeptide is connected to the Fc domain via a peptide linker.Docket No. 56146-746.601

[0042] 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.INCORPORATION BY REFERENCE

[0043] 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 the 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

[0044] FIG. 1A shows an exemplary embodiment of a multifunctional immunocytokine as described herein, which comprises an IL-2 polypeptide conjugated to an Fc domain, said Fc domain linked to two Fabs targeting VEGF and two scFvs targeting PD-1.

[0045] FIG. IB shows an analogous multifunctional immunocytokine to that of FIG. 1 A, but with an activatable IL-2 polypeptide depicted with its mask intact. The mask is linked to the IL-2 polypeptide by a protease cleavable linker. Upon cleavage of the protease cleavable linker, the mask is able to dissociate, thus allowing the IL-2 polypeptide to bind with the IL-2 receptor and signal.

[0046] FIGS. 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-2 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).

[0047] FIGS. 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 asDocket No. 56146-746.601 an IL-2 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: 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).

[0048] FIGS. 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-2 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).

[0049] FIGS. 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 canDocket No. 56146-746.601 be conjugated with a cytokine such as an IL-2 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, 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.

[0050] FIGS. 6A-6C depict formats of immunocytokine compositions according to the instant disclosure in which an IL-2 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.

[0051] FIGS. 7A-7I depict formats of immunocytokine compositions according to the instant disclosure which utilize a mask to block activity of the IL-2 polypeptide. Upon cleavage of the cleavable linker, the mask dissociates and renders the IL-2 polypeptide available for signaling with its receptor. In FIGs. 7A-7F, the mask (depicted as an anti-IL-2 scFv) is fused to the IL-2 polypeptide through the cleavable linker. In FIGs. 7G, 7H and 71, the mask is fused to the opposite Fc domain as the IL-2 polypeptide. 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.

[0052] FIGs. 8A-8H depict formats of immunocytokine compositions according to the instant disclosure which incorporate single domain antibodies specific for one of VEGFA or PD-1 into the composition.

[0053] FIGs. 9A-9H show dynamic light scattering (DLS) profiles of immunocytokine compositions described herein in the presence or absence of VEGF (left panels) and Analytical SEC-HPLC chromatogram of purified compositions with and without VEGF addition (detection: 220 nm) (right panels). FIG. 9A shows results for Composition 116. FIG. 9B shows results for Composition 117. FIG. 9C shows results for Composition 118. FIG. 9D shows results for Composition 119. FIG. 9E shows results for Composition 91. FIG. 9F shows results for Composition 98. FIG. 9G shows results for Composition 94. FIG. 9H shows results for Composition 120.

[0054] FIGs. 10A and 10B show the results of IL-2R HEKBlue® reporter assays for various immunocytokine compositions described herein in the presence or absence of VEGF.Docket No. 56146-746.601

[0055] FIGs. 11A and 11B show STAT5 induction of parental PDF NK92 cells and NK92 cells engineered to express human PD-1 by the indicated immunocytokines.

[0056] FIGs 12A and 12B show results of PD-1 / PD-L1 blocking assays using immunocytokine composition described herein.

[0057] FIG. 13A-13B shows results of VEGFR blocking assays using immunocytokine compositions described herein.

[0058] FIG. 14 shows STAT5 induction of PBMC subpopulations by immunocytokine compositions described herein.

[0059] FIG. 15 shows PK results from mice dosed with the indicated immunocytokines.

[0060] FIG. 16 shows average tumor growth curves, body weight change, and survival curves in an MC38 bearing C57BI / 6 mouse model transgenic for human PD-1 after administration of immunocytokine Composition 116.

[0061] FIG. 17 shows average tumor growth curves, body weight change, and survival curves in an MC38 bearing C57BI / 6 mouse model transgenic for human PD-1 after administration of immunocytokine Composition 117.

[0062] FIG. 18 shows body weight change and average tumor volume in an MKN45 tumor cell model in BalbC / nude mice administered one of Composition 117, Composition 118, or Composition 119.

[0063] While certain figures discussed supra include immunocytokine compositions or Fc domain contains scaffolds depicted as having various modifications to the Fc domain, compositions with alternative Fc domains or modifications thereof are also within the scope of the instant disclosure. The depiction of the formats with the indicated Fc modifications is not limiting.DETAILED DESCRIPTION

[0064] 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.

[0065] 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 theDocket No. 56146-746.601 context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment.

[0066] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.Multifunctional Immunocytokines

[0067] 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-2.Binding Domains

[0068] 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.

[0069] 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.

[0070] 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).

[0071] 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.Docket No. 56146-746.601

[0072] 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 DualAffinity 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).

[0073] 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, a tetrabody, a maxibody, a camelid, 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 camelid, a single domainDocket No. 56146-746.601 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, a bispecific F(ab')2, a variable fragment (Fv), a single chain variable fragment (scFv), disulfide stabilized Fv (dsFv), a camelid, or 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).

[0074] 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 a bispecific antibody. In some embodiments, the VH is comprised in a Fab, a Fab’, an scFv, or a VHH. In some embodiments, the VH is 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.

[0075] 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 toDocket No. 56146-746.601 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, L11R, 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 preexisting 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, 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).

[0076] 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, a variable fragment (Fv), a single chain variable fragment (scFv), a bispecificDocket No. 56146-746.601 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.

[0077] In some embodiments, a binding domain of the instant disclosure is a light chain single domain antibody.

[0078] 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

[0079] 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).

[0080] 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 receptorDocket No. 56146-746.601 mediated proliferation, and immune evasion by the cancerous cells. A non-limiting, exemplary, human PD-1 amino acid sequence isMQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLWTEGDNATFTCSF SNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVR ARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV VGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGEL DFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHC SWPL (SEQ ID NO: 31).

[0081] 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.

[0082] 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.

[0083] 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,Docket No. 56146-746.601CBT-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, Tebotelimab, 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.

[0084] 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, Spartalizumab, Cetrelimab, Tebotelimab, Cadonilimab,Docket No. 56146-746.601Pidilizumab, 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.

[0085] 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 1 A, 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 1 A.

[0086] 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.Docket No. 56146-746.601In 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.

[0087] TABLES 1 A and IB 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 described in Table 1A or IB are incorporated into a binding domain as a VHH. In some embodiments, a VH as described in Table 1 A or IB is incorporated into a binding domain as a VHH.

[0088] 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.

[0089] 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, 372, 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.

[0090] 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.

[0091] 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 inDocket No. 56146-746.601SEQ 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 acid 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, anDocket No. 56146-746.601 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.

[0092] 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).

[0093] 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).

[0094] 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 aminoDocket No. 56146-746.601 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).

[0095] 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 antibody (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0096] 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).

[0097] 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 1 A or IB. In some embodiments, the binding domain specific for VEGFA fused to the antibody of Table 1 A or IB is an anti- VEGFA single-domain antibody as described herein.Docket No. 56146-746.601

[0098] 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.

[0099] 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).

[0100] 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: 5 (e.g., the anti-PD-1 binding domain comprises the indicated sequence identity to SEQ ID NO: 5 and retains the CDRs).

[0101] 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).

[0102] 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).

[0103] 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 comprisedDocket No. 56146-746.601 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).

[0104] 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.TABLE 1A - Exemplary Antibodies Targeting PD-1 From Which anti-PD-1 binding domains can be derivedDocket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Table IBDocket No. 56146-746.601

[0105] 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 74, 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 74, 62, 70, 76, 84, or 178). In some embodiments, the anti-PD-1 binding domain is one of the VHHs provided in Table 1C. sequence (e.g., VHH 74, 62, 70, 76, 84, or 178).Table 1C - Exemplary anti-PD-1 VHH binding domainsDocket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601

[0106] 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), US20160207981 Al (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, L11R, LI ID, or LI IE substitution), US20140161796A1 (e.g., deletions of certainDocket No. 56146-746.601 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. 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).

[0107] 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 L154 of the light chain constant region (EU numbering), such as an LI 54K 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

[0108] 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 the disclosure specifically binds to VEGFA. In some embodiments, the antiDocket No. 56146-746.601VEGFA 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.

[0109] A non-limiting, exemplary, human VEGFA amino acid sequence is LTDRQTDTAPSPSYHLLPGRRRTVDAAASRGQGPEPAPGGGVEGVGARGVALKLFV QLLGCSRFGGAVVRAGEAEPSGAARSASSGREEPQPEEGEEEEEKEEERGPQWRLGA RKPGSWTGEAAVCADSAPAARAPQALARASGRGGRVARRGAEESGPPHSPSRRGSA SRAGPGRASETMNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKF MDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNI TMQIMRIKPHQGQHIGEMSFLQHNKCECRCDKPRR (SEQ ID NO: 120) (UniProt ID A0A0A0MR43). VEGFA is also referred to VEGF. The terms “VEGFA” and “VEGF” are used interchangeably herein.

[0110] 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.[OHl] 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, or those of an antibody or antigen binding fragment otherwise known in the art.

[0112] In one embodiment, an anti- VEGFA binding domain of the disclosure comprises the CDRs of an antibody selected from Bevacizumab, Brolucizumab, Faricimab, Ranibizumab,Docket No. 56146-746.601Ivonescimab, AI-081, HLX-04, or IBI305 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. 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.

[0113] 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 IBB 05. 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.Docket No. 56146-746.601

[0114] 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. In some embodiments, the anti- VEGFA binding domain comprises the VH and VL of Bevacizumab in a Fab or scFv format.

[0115] TABLES 2A and 2B provide the sequences of exemplary anti- EGFA 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 2A 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 2A or 2B is incorporated into a binding domain as a VHH.

[0116] 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.

[0117] 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 2A or 2B. In preferred embodiments, the VH and VL are from the same antibody or antigen binding fragment described in Table 2 A.

[0118] 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 2A or 2B.Docket No. 56146-746.601

[0119] In some embodiments, an anti-VEGFA binding domain comprises a VH having an amino acid sequence shown in Table 2 A, 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 2A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of Ivonescimab as shown in Table 2 A. In some embodiments, an anti-VEGFA binding domain comprises a VH and VL of AL081 as shown in Table 2 A. 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 2 A.

[0120] 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 2A 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).

[0121] 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 2 A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0122] 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 ofDocket No. 56146-746.601Brolucizumab as shown in Table 2A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0123] 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).

[0124] 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).

[0125] 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 2 A (e.g., contains the indicated CDRs and the VH and VL each comprise the indicated sequence identity overall).

[0126] In some embodiments, an anti-VEGFA binding domain comprises a VH CDR1 a VH CDR2 and a VH CDR3 of AL081 as shown in Table 2A comprised in a VH and a VL CDR1, a VL CDR2, and a VL CDR3 of AL081 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).

[0127] 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%,Docket No. 56146-746.60185%, 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).

[0128] 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 IB 1305 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).

[0129] 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).

[0130] 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).

[0131] 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).

[0132] 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 comprisedDocket No. 56146-746.601 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).

[0133] 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).

[0134] 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).

[0135] 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).

[0136] 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 2A. 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.Docket No. 56146-746.601

[0137] 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.TABLE 2A - Exemplary Antibodies Targeting VEGFA From Which anti-VEGFABinding Domains Can be DerivedDocket No. 56146-746.601Docket No. 56146-746.601VH CDR3: YPYYYGTSHWYFDV (SEQ ID NO: 125)Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Table 2B - Single Domain anti-VEGFA AntibodiesDocket No. 56146-746.601Docket No. 56146-746.601

[0138] 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. In some embodiments, the C-terminal “PP” of the VHH in Table 2 C can be omitted.Table 2C - Exemplary anti-VEGFA VHH binding domainsDocket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601

[0139] 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), US20160207981 Al (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, L11R, 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. 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).Docket No. 56146-746.601In 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).

[0140] 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).

[0141] 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 sequence DGGGIRRSMSGTWYLKAMTVDREFPEMNLESVTPMTLTLLKGHNLEAKVTMLISGR CQEVKAVLGRTKERKKYTADGGKHVAYIIPSAVRDHVIFYSEGQLHGKPVRGVKLV GRDPKNNLEALEDFEKAAGRLSTESILIPRQSETCSPG (SEQ ID NO: 226). In some embodiments, the anti-VEGFA binding domain comprises the sequence of SEQ ID NO: 226.Cytokines

[0142] 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 exhibitDocket No. 56146-746.601 enhanced biological activity compared to individual cytokines by themselves or can modulate the immune system in advantageous ways difficult to achieve with individual cytokines.

[0143] 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-lp, 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.

[0144] 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, half-life 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-2 Polypeptides

[0145] The present disclosure describes in some embodiments anti-VEGFA binding domains and / or anti-PD-1 binding domains linked to interleukin-2 (IL-2) polypeptides asDocket No. 56146-746.601 immunocytokine compositions and their use as human therapeutic agents. In some embodiments, the IL-2 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. Unless otherwise specified, residue position numbering in reference to a modification to an IL-2 polypeptide described herein refers to SEQ ID NO: 701 as a reference sequence.

[0146] In some embodiments, the IL-2 polypeptide is biased towards one or more of the IL-2 receptor subunits relative to WT IL-2. In some embodiments, the IL-2 polypeptide retains binding to the IL-2 receptor alpha subunit but has detuned binding for the IL-2 receptor beta and gamma subunits. Non-limiting examples of such IL-2 polypeptides include those found in US20230303649A1, any one of which can be used as an IL-2 polypeptide in an immunocytokine composition as described herein (or can be suitably modified to be incorporated into an immunocytokine composition as described herein).

[0147] In other embodiments, the IL-2 polypeptide retains binding to the IL-2 receptor beta and / or gamma subunits (or the beta gamma heterodimer subunit) buy lacks or exhibits substantially diminished ability to bind to the IL-2 receptor alpha subunit. Non-limiting examples of such IL-2 polypeptides include those found in US11633488B2, any one of which can be used as an IL-2 polypeptide in an immunocytokine composition as described herein (or can be suitably modified to be incorporated into an immunocytokine composition as described herein).

[0148] In some embodiments, the IL-2 polypeptide comprises a modification which imparts favorable properties related to stability and / or expression of the IL-2 polypeptide but which otherwise generally does not substantially impact the activity of the IL-2 polypeptide. Several such modifications include, for example, a deletion of residue Al of the IL-2 polypeptide, a T3A substitution, a C125S substitution, and / or a C125A substitution. Such modifications can generally be combined with other modifications described herein which can impact the activity of the IL-2 polypeptide described herein without causing detrimental effects. Thus, it is expressly contemplated within the instant disclosure that any IL-2 polypeptide described herein can further comprise a deletion of residue Al, a T3A substation, and / or a C125S or C125A substitution.

[0149] In some instances, it is desirable that the IL-2 polypeptide is one which is either activatable (e.g., has a null or low IL-2 related activity until activation, such as cleavage of a masking group by a tumor microenvironment protease) or one which is detuned (i.e., less potent / active) as compared to wild type IL-2 in order to provide a high degree of safety andDocket No. 56146-746.601 low toxicity / side effects. Non-limiting examples of such activatable IL-2 polypeptides can be found in WO2024150175A1, any one of which can be used as an IL-2 polypeptide in an immunocytokine composition as described herein (or can be suitably modified to be incorporated into an immunocytokine composition as described herein). Additionally, the activation strategies described therein could also be applied to other IL-2 polypeptides, such as those described below which favor binding to the IL-2 receptor alpha subunit.Alpha-competent IL-2 polypeptides with detuned IL-2 receptor beta / gamma subunit activity

[0150] In some embodiments, an IL-2 polypeptide of an immunocytokine composition described herein exhibits binding to and / or signaling through the IL-2 receptor alpha subunit (e.g., the IL-2 receptor aPy complex). In some embodiments, the IL-2 polypeptide exhibits binding to and / or signaling through the IL-2 receptor alpha subunit which is comparable to or only slightly diminished compared to wild type IL-2. In some embodiments, the IL-2 polypeptide contains substantially detuned ability to bind to and / or signal through the IL-2 receptor beta and / or gamma subunits.

[0151] In some embodiments, the IL-2 polypeptide comprises one or more modification which reduces binding of the IL-2 polypeptide to the IL-2 receptor beta subunit.

[0152] In some embodiments, the IL-2 polypeptide comprises a substitution at one or more residues selected from Hl 6, D20, D84, S87, N88, and V91. In some embodiments, the IL-2 polypeptide comprises one or more substitutions selected from H16S, D20V, D84K, S87A, N88D, N88R, V91A, and V91L. In some embodiments, the IL-2 polypeptide comprises an N88D or N88R substitution. In some embodiments, the IL-2 polypeptide comprises an amino acid substitution at residue N88. In some embodiments, the IL-2 polypeptide comprises an N88D substitution. In some embodiments, the IL-2 polypeptide comprises an N88R substitution.

[0153] In some embodiments, the IL-2 polypeptide comprises one or more modifications which reduce binding to the IL-2 receptor gamma subunit.

[0154] In some embodiments, the IL-2 polypeptide comprises a substitution at one or more residues selected from L12, E15, L19, Q22, T123, Q126, 1129, or S130. In some embodiments, the IL-2 polypeptide one or more substitution selected from L12A, L12V, L12Y, E15D, E15S, L19D, L19A, L19V, Q22T, T123A, Q126T, I129A, I129K, I129L or S130R. In some embodiments, the IL-2 polypeptide comprises a substitution at any one of residues L12, E15, L19, T123, Q126, or 1129. In some embodiments, the IL-2 polypeptide comprises one or more substitutions selected from L12A, L12Y, E15D, E15S, L19A, L19D, T123A, Q126T, I129A,Docket No. 56146-746.601 and I129K. In some embodiments, the IL-2 polypeptide comprises any one of the following sets of substitutions: Q126T; I129K; I129A, E15S, and T123A; E15D; L12A, L19A, and E15S; L12Y and L19D; L12A and L19A; or L19D. In some embodiments, the IL-2 polypeptide comprises an E15D or an L19D substitution. In some embodiments, the IL-2 polypeptide comprises an E15D substitution. In some embodiments, the IL-2 polypeptide comprises an L19D substitution.

[0155] In some embodiments, the IL-2 polypeptide comprises one or more modifications which favorably impact expression and / or stability of the IL-2 polypeptide.

[0156] In some embodiments, the IL-2 polypeptide comprises an Al deletion, a T3A substitution, a C125S substitution, or a C125A substitution. In some embodiments, the IL-2 polypeptide comprises a T3A substitution, a C125S substitution, or a C125A substitution. In some embodiments, the IL-2 polypeptide comprises a T3 A substitution. In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the IL-2 polypeptide a C125A substitution. In some embodiments, the IL-2 polypeptide comprises a T3A substitution and a C125A or C125S substitution. In some embodiments, the IL-2 polypeptide comprises a T3A substitution and a C125S substitution.

[0157] In some embodiments, the IL-2 exhibits reduced binding to heparin. In some embodiments, the IL-2 polypeptide comprises one or more modifications which reduce the binding of the IL-2 polypeptide to heparin. In some embodiments, reducing binding to heparin can favorably impact pharmacokinetic properties in vivo.

[0158] In some embodiments, the IL-2 polypeptide of comprises a modified B’C’ loop region of the IL-2 polypeptide. The B’C’ loop region refers to the amino acids which form the linkage between helixes B and C of IL-2 (e.g., human IL-2). The B’C’ loop region contains the amino acids positioned between amino acids 73 and 84 of wild type human IL-2 (SEQ ID NO: 701). In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide and insertion of an exogenous peptide into the B’C’ loop region. In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide and an insertion of an exogenous peptide into the B’C’ loop region.

[0159] In some embodiments, the modified B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acids 73 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region comprises a deletion of each amino acid 73Docket No. 56146-746.601 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region comprises a deletion of each amino acid between 73 and 84 of the IL-2 polypeptide and insertion of an exogenous peptide.

[0160] In some embodiments, the modified B’C’ loop region comprises insertion of an exogenous peptide. In some embodiments, the exogenous peptide comprises the sequence GDGSIN (SEQ ID NO: 700). In some embodiments, the exogenous peptide consists of the sequence GDGSIN (SEQ ID NO: 700). In some embodiments, the modified B’C’ loop region comprises a deletion of each amino acid between amino acids 73 and 84 of the IL-2 polypeptide and an insertion of an exogenous peptide having the sequence GDGSIN (SEQ ID NO: 700) (i.e., the amino acids between 73 and 84 of the IL-2 polypeptide are replaced with the sequence GDGSIN (SEQ ID NO: 700)). In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-72 of SEQ ID NO: 701 (i.e., the sequence APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQ CLEEELKPLEEVLNLA (SEQ ID NO: 784)) and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 701 (i.e., the sequenceDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 785). In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequence APTS S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKF YMPKKATELKHLQ CLEEELKPLEEVLNLAGDGSINDLISNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT (SEQ ID NO: 783). In some embodiments, the inserted exogenous peptide is QDASIH (SEQ ID NO: 786).

[0161] In some embodiments, the IL-2 polypeptide comprises a substitution at one or more amino acids selected from residue 32, residue 35, residue 38, residue 76, residue 81, or residue 83. In some embodiments, the IL-2 polypeptide comprises aK32S, K35E, R38A, K76A, R81S, or R83S substitution. In some embodiments, the IL-2 polypeptide comprises K32S. In some embodiments, the IL-2 polypeptide comprises K35E. In some embodiments, the IL-2 polypeptide comprises R38A. In some embodiments, the IL-2 polypeptide comprises K76A. In some embodiments, the IL-2 polypeptide comprises R81S. In some embodiments, the IL-2 polypeptide comprises R83S. In some embodiments, the IL-2 polypeptide comprises K32S, K35E, and R38A substitutions. In some embodiments, the IL-2 polypeptide comprises K76ADocket No. 56146-746.601 and R81S substitutions. In some embodiments, the IL-2 polypeptide comprises K76A, R81S, and R81S substitutions.

[0162] In some embodiments, the IL-2 polypeptide is one which 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: 704-774. In some embodiments, the IL-2 polypeptide is one which 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: 704-774 and retains all of the substitutions relative to the IL-2 polypeptide of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide is one which comprises the amino acid sequence set forth in any one of SEQ ID NOs: 704-774. In some embodiments, the IL-2 polypeptide is one which comprises the amino acid sequence set forth in SEQ ID NO: 751. In some embodiments, the IL-2 polypeptide is one which comprises the amino acid sequence set forth in SEQ ID NO: 753. In some embodiments, the IL-2 polypeptide is one which comprises the amino acid sequence set forth in SEQ ID NO: 754. In some embodiments, the IL-2 polypeptide is one which comprises the amino acid sequence set forth in SEQ ID NO: 758.

[0163] In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, F42A, Y45A, L72G, and C125A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, Y31H, K35R, Q74P, N88D, and C125A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88R, S130R, and IL15B'C' loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88R, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, D20V, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, H16S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, D20V, V91A, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, Q22T, V91L, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, Q22T, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, C125S, and Q126T. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, C125S, and I129A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, C125S, and I129L. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S,Docket No. 56146-746.601N88D, C125S, and I129A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, N88D, C125S, and I129L. In some embodiments, the IL- 2 polypeptide comprises the following substitutions: T3A, N88D, C125S, and I129K. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, H16S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, Q22T, N88D, E95S, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, Q22T, S87A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, D84K, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, Q22T, S87A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, N88D, C125S, and Q126T. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88R, C125S, and S130R. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, F42A, N88R, C125S, S130R, and IL15B'C' loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88R, C125S, S130R, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K76A, R81S, N88R, C125S, and S130R. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K76A, R81S, R83S, N88R, C125S, and S130R. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88R, C125S, S130R, and QSGH AB loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K32S, K35E, R38A, N88R, C125S, and S130R. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3 A, N88R, I92L, C125S, and S130R. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, N88D, T123A, C125S, and 1129 A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, T123A, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19A, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, E15S, L19A, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, L19D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15D, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S,Docket No. 56146-746.601L19D, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, E15S, L19A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, L19D, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12V, L19V, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, L19A, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L19D, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, Y31H, K35R, Q74P, N88D, C125A, and Q126T. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, C125S, Q126T, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, N88D, C125S, I129K, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, N88D, T123A, C125S, I129A, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15D, N88D, C125S, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19A, N88D, C125S, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, L19D, N88D, C125S, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, L19A, N88D, C125S, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L19D, N88D, C125S, and GDGSIN B'C loop. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K76A, R81S, N88D, C125S, and Q126T. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K76A, R81S, N88D, C125S, and I129K. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, K76A, R81S, N88D, T123A, C125S, and I129A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15D, K76A, R81S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19A, K76A, R81S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, L19D, K76A, R81S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, L19A, K76A, R81S, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L19D, K76A, R81S, N88D, and C125S. In some embodiments, the IL-2Docket No. 56146-746.601 polypeptide comprises the following substitutions: T3A, K32S, K35E, N88D, C125S, and Q126T. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, K32S, K35E, N88D, C125S, and I129K. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15S, K32S, K35E, N88D, T123A, C125S, and I129A. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, E15D, K32S, K35E, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, E15S, L19A, K32S, K35E, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12Y, L19D, K32S, K35E, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L12A, L19A, K32S, K35E, N88D, and C125S. In some embodiments, the IL-2 polypeptide comprises the following substitutions: T3A, L19D, K32S, K35E, N88D, and C125S.

[0164] Non-limiting examples of IL-2 polypeptides of the instant disclosure include those listed in Table 3 below. In Table 3 and the preceding paragraph,, “IL15B'C loop” refers to a deletion of each amino acid between residues 73 and 84 of the IL-2 polypeptide (SEQ ID NO: 701 as a reference sequence) and insertion of the sequence GDASH4 (SEQ ID NO: 786). In Table 3 and the preceding paragraph, “GDGSIN B'C loop” refers to a deletion of each amino acid between 73 and 84 of the IL-2 polypeptide (SEQ ID NO: 701 as a reference sequence) and insertion of the sequence GDGSIN (SEQ ID NO: 700). In Table 3 and the preceding paragraph, “QSGH AB loop” refers to a deletion of each amino acid between residues 28 and 39 (SEQ ID NO: 701 as a reference sequence) and insertion of the sequence QSGH (SEQ ID NO: 787).Table 3 - Exemplary IL-2 PolypeptidesDocket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Beta-gamma competent IL-2 polypeptides with detuned IL-2 receptor alpha binding

[0165] In some embodiments, an IL-2 polypeptide of an immunocytokine composition described herein is biased in favor of binding to and / or signaling through the IL-2 receptor beta subunit, IL-2 receptor gamma subunit, or the IL-2 receptor beta / gamma complex. In some embodiments, such IL-2 polypeptides exhibits substantially reduced binding to IL-2 receptor alpha subunit or the receptor alpha / beta / gamma complex. Non-limiting examples of such IL-2Docket No. 56146-746.601 polypeptides are described in, for example, US11633488B2, the contents of which are incorporated by reference. In some embodiments, an IL-2 polypeptide described therein is preferred for use in the immunocytokine composition of the instant disclosure (e.g., Composition A2 shown in FIG. 1H therein).

[0166] In some preferred embodiments of immunocytokine compositions which contain IL-2 polypeptides which exhibit substantially no ability to bind the IL-2 receptor alpha subunit yet retain binding and signaling ability through the beta and / or gamma subunit, the IL-2 polypeptide comprises polymers (e.g., PEG polymers) attached at one or both of residues 42 and 45 of the IL-2 polypeptide (e.g., F42Y and Y45). In some embodiments, one of the polymers forms a part of the linker which attaches the IL-2 polypeptide to the immunocytokine composition (e.g., the polymer attached at residue F42Y contains a conjugation handle such as an azide). In some embodiments, such an IL-2 polypeptide is synthetic (e.g., synthesized via KAHA ligation and further containing Hse41, Hse71, Hsel04, Nle23, Nle39, and Nle 46). In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the polymers (E.g., the PEG polymers) attached at these residues 42 and 45 have a molecular weight of about 200-1000 Daltons. In some embodiments, such an IL-2 polypeptide comprises SEQ ID NO: 703.

[0167] Additional IL-2 polypeptides with similar properties are also contemplated as within the scope of the instant disclosure. Modifications to such IL-2 polypeptides 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.

[0168] In some embodiments, the IL-2 polypeptide of the comprises one or more modifications in addition to a modification needed to attach the linker to the relevant residue of the IL-2 polypeptide e.g., an amino acid substitution at a residue to which the linker is not attached).

[0169] In some embodiments, the IL-2 polypeptide of the immunocytokine composition described herein contains 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.Docket No. 56146-746.601

[0170] In some embodiments, the IL-2 polypeptide of the immunocytokine composition 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.

[0171] In some embodiments, the IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 703. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 703. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 703.

[0172] In some embodiments, the IL-2 polypeptide of the immunocytokine composition 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-2 polypeptide comprises 1 to 9 amino acid substitutions. In some embodiments, the IL-2 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 acid 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-2 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-2 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.

[0173] In some embodiments, the IL-2 polypeptide comprising of the immunocytokine composition 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 natural amino acid substitutions, wherein the natural amino acid substitutions are relative to SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises 1 to 9 natural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises 1 or 2 natural amino acid substitutions, 1 to 3 natural amino acid substitutions, 1 toDocket No. 56146-746.6014 natural amino acid substitutions, 1 to 5 natural amino acid substitutions, 1 to 6 natural amino acid substitutions, 1 to 7 natural amino acid substitutions, 1 to 8 natural amino acid substitutions, 2 to 3 natural amino acid substitutions, 2 to 4 natural amino acid substitutions, 2 to 5 natural amino acid substitutions, 2 to 6 natural amino acid substitutions, 2 to 7 natural amino acid substitutions, 2 to 8 natural amino acid substitutions, 2 to 9 natural amino acid substitutions, 3 or 4 natural amino acid substitutions, 3 to 5 natural amino acid substitutions, 3 to 6 natural amino acid substitutions, 3 to 7 natural amino acid substitutions, 3 to 9 natural amino acid substitutions, 4 or 5 natural amino acid substitutions, 4 to 6 natural amino acid substitutions, 4 to 7 amino acid substitutions, 4 to 9 natural amino acid substitutions, 5 or 6 natural amino acid substitutions, 5 to 7 amino acid substitutions, 5 to 9 natural amino acid substitutions, 6 or 7 natural amino acid substitutions, 6 to 9 natural amino acid substitutions, or 7 to 9 natural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises 3 natural amino acid substitutions, 4 natural amino acid substitutions, 5 amino acid substitutions, 6 natural amino acid substitutions, 7 natural amino acid substitutions, or 9 natural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises at most 4 natural amino acid substitutions, 5 natural amino acid substitutions, 6 natural amino acid substitutions, 7 natural amino acid substitutions, or 9 natural amino acid substitutions. In some embodiments, the IL-2 polypeptide further comprises up to 10 non-canonical amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 unnatural amino acid substitutions. In some embodiments, the IL-2 polypeptide further comprises unnatural amino acid substitutions at residues M23, M39, and / or M46. In some embodiments, the unnatural amino acid residues substituted for the methionines are each independently norleucine or O-methyl-homoserine. In some embodiments, the IL-2 polypeptide further comprises unnatural amino acid substitutions at residues 41, 71, and 104. In some embodiments, the IL-2 polypeptide further comprises homoserine (Hse) 41, Hse 71, and Hse 104.

[0174] In some embodiments, the IL-2 polypeptide comprises at least one substitution or modification (e.g., attachment of a polymer) to the amino acid sequence of SEQ ID NO: 701. In some embodiments, the at least one substitution or modification has an impact on the ability of the IL-2 polypeptide to bind to one or more IL-2 receptor subunits. In some embodiments, the at least one substitution or modification diminishes the ability of the IL-2 polypeptide to bind to the IL-2 receptor a subunit. Further non-limiting examples such modifications are described in, for example, PCT Publication Nos. WO2021140416A2, W02012065086A1,Docket No. 56146-746.601WO20 19028419A1, W02012107417A1, WO2018119114A1, WO2012062228 A2, W02019104092A1, WO2012088446A1, and WO2015164815A1, each of which is hereby incorporated by reference as if set forth herein in its entirety. In addition to modifications of IL-2 which may affect binding to one or more IL-2 receptor subunits (such as the alpha subunit), the IL-2 polypeptide provided herein may also comprises one or more modifications which improve the stability or pharmacokinetic properties of the IL-2 polypeptide. For example, the IL-2 polypeptide provided herein can comprise the modifications relative to SEQ NO: 701 which are contained in aldesluekin (Proleukin®) (SEQ ID NO: 702), namely a deletion of the N-terminal A residue of WT IL-2 and a C125S substitution relative to WT IL- 2.

[0175] Non-limiting examples of modifications to IL-2 polypeptides include amino acid substitutions shown in Table 4 or 5 below. In some embodiments, the IL-2 polypeptide comprises 1, 2, 3, 4, 5, or more of the amino acid substitutions set forth in the Tables 4 and 5 below.Table 4 - IL-2 Substitutionable 5 - Additional IL-2 SubstitutionsDocket No. 56146-746.601

[0176] In some embodiments, the IL-2 polypeptide comprises at least one modification is in the range of amino acid residues 30-75. In some embodiments, the IL-2 polypeptide comprises at least one polymer attachment to the residue at position 42 and / or 45 and / or an amino acid substitution at residue position 42 and / or 45. In some embodiments, one modification is at amino acid residue 42. In some embodiments, one modification is aF42Y substitution. In some embodiments, one modification is a polymer attached to residue F42Y. In some embodiments, one modification is at residue 45. In some embodiments, the modification at residue 45 is a polymer attached to residue 45. In some embodiments, the modification at residue 45 is a polymer attached to residue Y45. In some embodiments, the IL-2 polypeptide comprises a first polymer attached at residue F42Y and a second polymer attached at residue Y45. In some embodiments, the IL-2 polypeptide comprises a deletion of residue 1 from SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the IL-2 polypeptide further comprises one or more substitutions of a synthetic IL-2 polypeptide as provided herein (e.g., Hse or Nle substitutions).

[0177] In one embodiment an IL-2 polypeptide of an immunocytokine composition as provided herein (e.g., with a linker attached to a residue as provided herein, such as the N- terminal residue), further comprising a first polymer covalently attached at residue 42 and a second polymer covalently attached at residue 45, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the first polymer and the second polymer are the same. In some embodiments, the first polymer and the second polymer are different. In some embodiments, each polymer is attached throughDocket No. 56146-746.601 a tyrosine residue. In some embodiments, the IL-2 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: 701. In some embodiments, the IL-2 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-2 polypeptide comprises a C125S or C125A substitution. In some embodiments, the IL-2 polypeptide comprises a deletion of Al from the sequence of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises amino acid substitutions at 1, 2, 3, or 4 methionine residues from SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide further comprises unnatural amino acid substitutions at residues M23, M39, and / or M46. In some embodiments, the unnatural amino acid residues substituted for the methionines are each independently norleucine or O-methyl-homoserine. In some embodiments, the IL-2 polypeptide further comprises homoserine Hse 41, Hse 71, and Hse 104.

[0178] 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 to SEQ ID NO: 703. In some embodiments, the IL-2 polypeptide is synthetic.

[0179] In some embodiments, the IL-2 receptor beta / gamma biased IL-2 polypeptide a polymer attached to a residue of the IL-2 polypeptide (e.g., a polymer in addition to the linker attached at the point of attachment). In some embodiments, the polymer is attached to a different residue than the residue to which the linker is attached.

[0180] In some embodiments, the polymer is attached to an amino acid residue of the IL-2 polypeptide. In some embodiments, the polymer is attached to any amino acid residue of the IL-2 polypeptide (e.g., at a position corresponding to any one of positions 1-133 of SEQ ID NO: 701). In some embodiments, the polymer is attached at a non-terminal residue (e.g., a residue other than the C-terminal residue or N-terminal residue) of the IL-2 polypeptide (e.g., a residue at position corresponding to any one of positions 2-132 of SEQ ID NO: 701). In some embodiments, the polymer is attached at a terminal residue of the IL-2 polypeptide, wherein the IL-2 polypeptide has been extended or truncated by one or more amino acids relative to SEQ ID NO: 701 (e.g., the linker is attached to a residue corresponding to residue 2 of SEQ ID NO: 701 and residue 1 of SEQ ID NO: 701 has been deleted). In some embodiments, the polymer is attached to the N-terminal residue of the IL-2 polypeptide. In some embodiments, the polymer is attached to the N-terminal amine of the IL-2 polypeptide. In some embodiments,Docket No. 56146-746.601 the polymer is attached to the C-terminal residue of the IL-2 polypeptide. In some embodiments, the polymer is attached to the C-terminal carboxyl group of the IL-2 polypeptide.

[0181] In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue in a region comprising residues 2-132, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue in a region comprising residues 30-75. In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue in a region comprising residues 35-55, residues 35-50, residues 35-45, residues 30-50, residues 40-45, residues 60-75, residues 60-70, residues 65-70, or residues 2-5. In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue selected from residue 65, 66, 67, 68, 69, and 70. In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue selected from residue 40, 41, 42, 43, 44, and 45. In some embodiments, the polymer is attached to the IL-2 polypeptide at residue 42 or 45. In some embodiments, the polymer is attached to the IL-2 polypeptide at residue 42. In some embodiments, the polymer is attached to the IL-2 polypeptide at residue 45.

[0182] In some embodiments, the polymer is attached to the IL-2 polypeptide at a residue which disrupts binding of the IL-2 polypeptide with the IL-2 receptor alpha subunit (TL-2Ra). Examples of these residues include residues 3, 5, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 65, 67, 68, 69, 71, 72, 103, 104, 105, and 107, as described in, for example, PCT Pub. Nos. WO2019028419A1, W02020056066A1, WO2021140416A2, and WO2021216478A1 each of which is hereby incorporated by reference as if set forth in its entirety. In some embodiments, the polymer is covalently attached at a residue selected from residues corresponding to residues 3, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63,64, 65, 67, 68, 69, 71, 72, 103, 104, 105, and 107 of SEQ ID NO: 701. In some embodiments, the polymer is covalently attached at residue 1, 35, 37, 38, 41, 42, 43, 44, 45, 60, 61, 62, 64,65, 68, 69, 71, 72, 104, 105, or 107 of the IL-2 polypeptide. In some embodiments, the polymer is covalently attached at residue 5. In some embodiments, the polymer is covalently attached at residue 38. In some embodiments, the polymer is covalently attached at residue 42. In some embodiments, the polymer is covalently attached at residue 45. In some embodiments, the polymer is covalently attached at residue 61. In some embodiments, the polymer is covalently attached at residue 65. In some embodiments, the polymer is covalently attached at residue 68.

[0183] In some embodiments, the residue to which the polymer is attached is a natural amino acid residue. In some embodiments, the residue to which the polymer is covalently attached is selected from cysteine, aspartate, asparagine, glutamate, glutamine, serine,Docket No. 56146-746.601 threonine, lysine, and tyrosine. In some embodiments, the residue to which the polymer is covalently attached is selected from asparagine, aspartic acid, cysteine, glutamic acid, glutamine, lysine, and tyrosine. In some embodiments, the polymer is covalently attached to a cysteine. In some embodiments, the polymer is covalently attached to a lysine. In some embodiments, the polymer is covalently attached to a glutamine. In some embodiments, the polymer is covalently attached to an asparagine. In some embodiments, the residue to which the polymer is attached is a tyrosine. In some embodiments, the residue to which the polymer is attached is the natural amino acid in that position in SEQ ID NO: 701 (e.g., Y45 or Al).

[0184] In some embodiments, the polymer 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 any naturally occurring amino acid, or any unnatural amino acid (e.g., an amino acid containing a desired reactive group, such as a CLICK chemistry reagent such as an azide, alkyne, etc. . In some embodiments, the 1 polymer is covalently attached to site-specifically to a natural amino acid.

[0185] In some embodiments, the polymer is attached to a tyrosine residue. In some embodiments, the polymer attached to the tyrosine residue has a structure:wherein n is 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. In some embodiments, the polymer attached to the tyrosine residue is at residue F42Y. In some embodiments, the polymer attached to the tyrosine residue is at Y45. In some embodiments, the IL-2 polypeptide comprises two polymers attached to tyrosine residues at F42Y and Y45. In some embodiments, the two polymers are the same size.Docket No. 56146-746.601

[0186] In some embodiments, the polymer 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 polymer to the IL-2 polypeptide. The conjugation handle can be any of the conjugation handles provided herein, and is preferably a different conjugation handle which is non-reactive with a conjugation handle used to attach or form part of the linker (where a conjugation handle is used to form the linker). In some embodiments, the polymer is covalently attached site-specifically to the unnatural amino acid. Non-limiting examples of amino acid residues comprising conjugation handles can be found, for example, in PCT Pub. Nos. WO2015054658A1, WO2014036492A1, and WO2021133839A1 W02006069246A2, and W02007079130A2, each of which is incorporated by reference as if set forth in its entirety. In some embodiments, the polymer is attached to an unnatural amino acid residue without use of a conjugation handle.

[0187] In some embodiments, the polymer is covalently attached at residue 42. In some embodiments, the polymer is covalently attached at residue F42E, F42D, F42Q, F42K, F42N, or F42Y. In some embodiments, the polymer is covalently attached at residue F42Y. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 42.

[0188] In some embodiments, the polymer is covalently attached at residue 45. In some embodiments, the polymer is covalently attached at residue Y45, Y45E, Y45C, Y45D, Y45Q, Y45K, or Y45N. In some embodiments, the polymer is covalently attached at residue Y45. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 45.

[0189] In some embodiments, the polymer is covalently attached at residue 65. In some embodiments, the polymer is covalently attached at residue P65C, P65D, P65Q, P65E, P65N, P65K, or P65Y. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 65.

[0190] In some embodiments, the polymer is covalently attached at residue 5. In some embodiments, the polymer is covalently attached at residue S5C, S5D, S5Q, S5K, S5N, S5K, or S5Y. In some embodiments, the polymer is covalently attached to an unnatural amino acid at residue 5.

[0191] In some embodiments, the polymer is covalently attached at residue 1. In some embodiments, the polymer is covalently attached at residue Al. In some embodiments, the polymer is covalently attached to the N-terminal amine of the IL-2 polypeptide.

[0192] In some embodiments, the polymer comprises a water-soluble polymer. In some embodiments, the water-soluble polymer comprises poly(alkylene oxide), polysaccharide,Docket No. 56146-746.601 poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, poly(acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer is poly(alkylene oxide). In some embodiments, the water-soluble polymer is polysaccharide. In some embodiments, the water-soluble polymer is poly(ethylene oxide) (PEG).

[0193] In some embodiments, the polymer has a molecular weight of from about 0.1 kDa to about 50 kDa. In some embodiments, the polymer has a molecular weight of from about 0.1 kDa to about 0.5 kDa from about 0.1 kDa to about 1 kDa, from about 0.1 kDa to about 2 kDa, from about 0.1 kDa to about 5 kDa from about 0.2 kDa to about 1 kDa, from about 0.2 kDa to about 2 kDa, from about 0.2 kDa to about 5 kDa, from about 0.2 kDa to about 10 kDa, from about 0.2 kDa to about 30 kDa, from about 0.5 kDa to about 2 kDa, from about 0.5 kDa to about 5 kDa, from about 0.5 kDa to about 10 kDa, from about 0.5 kDa to about 30 kDa, from about 1 kDa to about 5 kDa, from about 1 kDa to about 10 kDa, from about 1 kDa to about 30 kDa, from about 1 kDa to about 50 kDa, from about 2 kDa to about 10 kDa, from about 2 kDa to about 30 kDa, from about 2 kDa to about 50 kDa, from about 5 kDa to about 30 kDa, or from about 5 kDa to about 50 kDa. In some embodiments, the polymer has a molecular weight of at least about 0.2 kDa, at least about 0.5 kDa, at least about 1 kDa, at least about 2 kDa, at least about 5 kDa, at least about 10 kDa, or at least about 30 kDa. In some embodiments, the polymer has a molecular weight of at most about 30 kDa, at most about 10 kDa, at most about 5 kDa, at most about 2 kDa, at most about 1 kDa, at most about 0.5 kDa or at most about 0.2 kDa. In some embodiments, the polymer has a molecular weight of about 0.5 kDa, about 1 kDa, about 2 kDa, about 3 kDa, about 4 kDa, about 5 kDa, about 7.5 kDa, about 10 kDa, about 12.5 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 KDa, about 40 kDa, about 45 kDa, or about 50 kDa. In some embodiments, the polymer is a PEG polymer.

[0194] In some embodiments, the polymer is linear. In some embodiments, the polymer is a linear PEG polymer. In some embodiments, the polymer is branched. In some embodiments, the polymer is a branched PEG polymer. In some embodiments, the branched PEG polymer comprises a plurality of PEG chains from a central source molecule (e.g., a lysine or polylysine source molecule). In some embodiments, the polymer comprises from 1 to 10 polyethylene glycol chains. In some embodiments, the polymer comprises 1 polyethylene glycol chains to 10 polyethylene glycol chains. In some embodiments, the polymer comprises 1 polyethylene glycol chains to 2 polyethylene glycol chains, 1 polyethylene glycol chains to 4 polyethylene glycol chains, 1 polyethylene glycol chains to 6 polyethylene glycol chains, 1 polyethylene glycol chains to 10 polyethylene glycol chains, 2 polyethylene glycol chains to 4 polyethylene glycol chains, 2 polyethylene glycol chains to 6 polyethylene glycol chains, 2Docket No. 56146-746.601 polyethylene glycol chains to 10 polyethylene glycol chains, 4 polyethylene glycol chains to 6 polyethylene glycol chains, 4 polyethylene glycol chains to 10 polyethylene glycol chains, or 6 polyethylene glycol chains to 10 polyethylene glycol chains. In some embodiments, the polymer comprises 1 polyethylene glycol chains, 2 polyethylene glycol chains, 4 polyethylene glycol chains, 6 polyethylene glycol chains, or 10 polyethylene glycol chains. In some embodiments, the first water-soluble polymer comprises at least 1 polyethylene glycol chains, 2 polyethylene glycol chains, 4 polyethylene glycol chains, or 6 polyethylene glycol chains. In some embodiments, the first water-soluble polymer comprises at most 2 polyethylene glycol chains, 4 polyethylene glycol chains, 6 polyethylene glycol chains, or 10 polyethylene glycol chains. In some embodiments, the polymer comprises 4 polyethylene glycol chains.

[0195] In some embodiments, the polymer is an end-capped polymer. In some embodiments, the polymer is an end-capped polyethylene glycol. In some embodiments, the polymer is endcapped with a functional group selected from amine, alkoxy (e.g., methoxy, ethoxy, propoxy, etc.), hydroxyl, amide (e.g., -NH(C=O)(CI-C4 alkyl), carboxylate, and ester (e.g., methyl ester, ethyl ester, etc.). In some embodiments, the polymer as an amine end-capped PEG.

[0196] In some embodiments, the IL-2 polypeptide comprises two polymers covalently attached to two separate residues of the IL-2 polypeptide. In some embodiments, the two polymers are a first polymer and a second polymer. Each of the first polymer and the second polymer can be attached to the IL-2 polypeptide at any of the residues as provided herein and can be any of the polymers provided herein (e.g., having any combination of sizes as provided herein). In some embodiments, both of the first polymer and the second polymer are the same size or about the same size. In some embodiments, both polymers are at most about 1 kDa. In some embodiments, one polymer is substantially larger than the other. In some embodiments, one polymer is at most about 1 kDa and the other polymer is at least about 5 kDa.

[0197] A non-limiting set of IL-2 polypeptides provided herein with various linker points of attachment and polymers as provided herein is shown in Table 6 below.Table 6 — Exemplary Polymer Attachment Sites to IL-2Docket No. 56146-746.601*Residue position numbering based on SEQ ID NO: 701 as a reference sequence

[0198] In some embodiments, the IL-2 polypeptide comprises the linker covalently attached to the N-terminus, a first polymer covalently attached at residue 42, and a second polymer covalently attached at residue 45. In some embodiments, the first polymer is covalently attached at residue F42Y. In some embodiments, the second polymer is covalently attached at residue Y45. In some embodiments, the first polymer and the second polymer are different sizes. In some embodiments, the first polymer has a molecular weight of at most about 1 kDa and the second polymer has a molecular weight of at least about 5 kDa. In some embodiments, the first polymer has a molecular weight of from about 0.1 kDa to about 1 kDa and the second polymer has a molecular weight of from about 5 kDa to about 50 kDa. In some embodiments, the first polymer has a molecular weight of at least about 5 kDa and the second polymer has a molecular weight of at most about 1 kDa. In some embodiments, the first polymer has a molecular weight of from about 5 kDa to about 50 kDa and the second polymer has a molecular weight of from about 0.1 kDa to about 1 kDa. In some embodiments, the first polymer and the second polymer are the same or about the same size. In some embodiments, the first polymer and the second polymer each have a molecular weight of from about 0.1 kDa to about 1 kDa, about 0.2 kDa to about 1 kDa, or from about 0.5 kDa to about 1 kDa.

[0199] In some embodiments, the IL-2 polypeptide comprises the linker covalently to residue 42, a first polymer covalently attached at residue 45, and a second polymer covalently attached at the N-terminus. In some embodiments, the linker is attached at residue F42Y. In some embodiments, the first polymer is covalently attached at residue Y45. In some embodiments, the first polymer and the second polymer are different sizes. In some embodiments, the first polymer has a molecular weight of at most about 1 kDa and the second polymer has a molecular weight of at least about 5 kDa. In some embodiments, the first polymer has a molecular weight of from about 0.1 kDa to about 1 kDa and the second polymer has a molecular weight of from about 5 kDa to about 50 kDa. In some embodiments, the first polymer has a molecular weight of at least about 5 kDa and the second polymer has a molecular weight of at most about 1 kDa. In some embodiments, the first polymer has a molecular weight of from about 5 kDa to about 50 kDa and the second polymer has a molecular weight of from about 0.1 kDa to about 1 kDa.Docket No. 56146-746.601In some embodiments, the first polymer and the second polymer are the same or about the same size. In some embodiments, the first polymer and the second polymer each have a molecular weight of from about 0.1 kDa to about 1 kDa, about 0.2 kDa to about 1 kDa, or from about 0.5 kDa to about 1 kDa.

[0200] In some embodiments, the IL-2 polypeptide comprises the linker covalently attached to residue 45, a first polymer covalently attached at residue 42, and a second polymer covalently attached at the N-terminus. In some embodiments, the first polymer is covalently attached at residue F42Y. In some embodiments, the linker is covalently attached at residue Y45. In some embodiments, the first polymer and the second polymer are different sizes. In some embodiments, the first polymer has a molecular weight of at most about 1 kDa and the second polymer has a molecular weight of at least about 5 kDa. In some embodiments, the first polymer has a molecular weight of from about 0.1 kDa to about 1 kDa and the second polymer has a molecular weight of from about 5 kDa to about 50 kDa. In some embodiments, the first polymer has a molecular weight of at least about 5 kDa and the second polymer has a molecular weight of at most about 1 kDa. In some embodiments, the first polymer has a molecular weight of from about 5 kDa to about 50 kDa and the second polymer has a molecular weight of from about 0.1 kDa to about 1 kDa. In some embodiments, the first polymer and the second polymer are the same or about the same size. In some embodiments, the first polymer and the second polymer each have a molecular weight of from about 0.1 kDa to about 1 kDa, about 0.2 kDa to about 1 kDa, or from about 0.5 kDa to about 1 kDa.

[0201] In some embodiments, the IL-2 polypeptide comprises the linker covalently attached to residue 45 and a polymer covalently attached at residue 42. In some embodiments, the linker is attached at residue Y45. In some embodiments, the polymer is attached at residue F42Y. In some embodiments, the polymer has a molecular weight of at most about 1 kDa. In some embodiments, the polymer has a molecular weight of from about 0.1 kDa to about 1 kDa. In some embodiments, the polymer has a molecular weight of at least about 5 kDa. In some embodiments, the polymer has a molecular weight of from about 5 kDa to about 50 kDa.

[0202] In some embodiments, the IL-2 polypeptide comprises the linker covalently attached to residue 42 and a polymer covalently attached at residue 45. In some embodiments, the linker is attached at residue F42Y. In some embodiments, the polymer is attached at residue Y45. In some embodiments, the polymer has a molecular weight of at most about 1 kDa. In some embodiments, the polymer has a molecular weight of from about 0.1 kDa to about 1 kDa. In some embodiments, the polymer has a molecular weight of at least about 5 kDa. In some embodiments, the polymer has a molecular weight of from about 5 kDa to about 50 kDa.Docket No. 56146-746.601

[0203] Exemplary IL-2 polypeptides to which polymers can be attached to provide IL-2 polypeptides with reduced alpha subunit binding and retention of beta / gamma subunit binding are shown in Table 4 below, as well as the sequence of WT IL-2 (SEQ ID NO: 701 of Table 7) and aldesleukin (SEQ ID NO: 702 of Table 7). In some embodiments, the IL-2 polypeptide of SEQ ID NO: 703 modified with polymers attached at residues F42Y and Y45 is used in an immunocytokine composition of the instant disclosure.Table 7 - Exemplary IL-2 Sequences

[0204] In the table above, Nle is a norleucine residue and Hse is a homoserine residue.

[0205] One exemplary IL-2 of the instant disclosure which can be used to form a conjugated immunocytokine described herein is referred to herein as “2P72.” 2P72, its manufacture, properties, and derivatives thereof are described in detail in U.S. Patent No. 11,633,488 (described therein as “Composition A2”). 2P72 has a base sequence as set forth in SEQ ID NO: 703 and contains polymers attached at residues F42Y and Y45 as depicted in the structures below:Docket No. 56146-746.601IL-2 Polypeptides Which Selectively Bind to IL-2 Receptor Alpha Subunit with Enhanced Binding to IL-2 Receptor Alpha Subunit

[0206] In some embodiments, an IL-2 polypeptide incorporated into an immunocytokine composition described herein is an IL-2 polypeptide which exhibits enhanced alpha subunit binding but lacks or has severely impaired binding to the IL-2 receptor beta and / or gamma subunits. Non-limiting examples of such IL-2 polypeptides can be found in US20230303649A1, the contents of which are incorporated herein by reference.

[0207] In some embodiments, a preferred IL-2 polypeptide having such characteristics is the IL-2 polypeptide of SEQ ID NO: 776 (e.g., an IL-2 polypeptide having Y31H, K35R, Q74P, N88D, C125S, Hse41, Hse71, Hsel04, Nle23, Nle39, and Nle46 substitutions relative to WT IL-2). In some embodiments, the IL-2 polypeptide is the IL-2 polypeptide described as “Composition A” in US20230303649A1 (e.g., an IL-2 polypeptide having SEQ ID NO: 776 and an azide conjugation handle attached to the N-terminus as shown therein). Additional IL- 2 polypeptides having similar properties are which are compatible with the instant disclosure are described in more detail below.

[0208] Such IL-2 polypeptides may display binding characteristics for the IL-2 receptor (IL- 2R) that differ from wild-type IL-2 (SEQ ID NO:701) or aldesleukin (SEQ ID NO: 702). In one aspect, IL-2 polypeptides described herein have increased affinity for the IL-2R a complex. In some embodiments, the IL-2 polypeptides have an unmodulated affinity for the IL-2R Py complex. In some embodiments, the IL-2 polypeptides have a reduced affinity for the IL-2R Py complex. In some embodiments, the IL-2 polypeptides provided herein may comprise amino acid substitutions that enhance the binding affinity for the IL-2Ra receptor subunit. In some embodiments, the IL-2 polypeptides provided herein comprise amino acid substitutions that lower the IL-2 polypeptides affinity for the IL-2RP receptor subunit. In some embodiments, the IL-2 polypeptides have a biological activity of inducing fewer T-effector (Teff) cells when administered in vivo compared to a wild type IL-2 or aldesleukin. In some embodiments, the IL-2 polypeptides provided herein have comparable ability (e.g., have anDocket No. 56146-746.601EC50 no more than lOx greater, no more than lOOx greater) to induce regulatory T-cells (Treg) when administered in vivo compared to a wild type IL-2 or aldesleukin.

[0209] In some embodiments, the IL-2 polypeptides described herein contain modified amino acid residues. Such modifications can take the form of amino acid substitutions of a wild type IL-2 polypeptide such as the amino acid sequence of SEQ ID NO: 701, addition or deletion of amino acids from the sequence of SEQ ID NO: 701, or the addition of moieties to amino acid residues. In some embodiments, the IL-2 polypeptide described herein contains a deletion of the first amino acid from the sequence of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide described herein comprises a C125S substitution, using the sequence of SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide described herein comprises substitutions at one or more residues selected from Y31, K35, Q74, and / or N88, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. These substitutions may be in combination with the C125S substitution and / or an N-terminal deletion, such as a deletion of the first amino acids from the sequence of SEQ ID NO: 701. In some embodiments, the Y31 substitution is a Y31H substitution. In some embodiments, the K35 substitutions is a K35R substitution. In some embodiments, the Q74 substitution is a Q74P substitutions. In some embodiments, the N88 substitution is an N88D substitution. In some embodiments, the IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, and a Q74P substitution. In some embodiments, the IL-2 polypeptide comprises a Y31H substitution, a K35R substitution, a Q74P substitution, and an N88D substitution. In some embodiments, the IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, and a C125S substitution. In some embodiments, the IL-2 polypeptide comprises a Y31H substitution, a K35S substitution, a Q74P substitution, a N88D substitution, and a C125S substitution.

[0210] In some embodiments, the IL-2 polypeptide is a synthetic polypeptide. In some embodiments, the IL-2 polypeptide is synthesized by a-ketoacid-hydroxylamine (KAHA) amide-forming ligation. In some embodiments, the IL-2 polypeptide comprises unnatural amino acids, such as homoserine, which are used during the KAHA ligation reaction to join multiple polypeptide fragments to synthesize the full-length IL-2 polypeptide. In some embodiments, these are the only unnatural amino acids in the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises norleucine (Nle) residue substitutions at one or more methionine residues present in wild type IL-2 or aldesleukin. In some embodiments, the IL-2 polypeptide comprises norleucine residues at positions 23, 39, and 46.Docket No. 56146-746.601

[0211] In some embodiments, the IL-2 polypeptide as described herein can comprise one or more non-canonical amino acids (also referred to herein as “unnatural amino acids”). “Non- canonical” amino acids can refer to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins. In some embodiments, one or more amino acids of the IL-2 polypeptides are substituted with one or more non-canonical amino acids. Non-canonical amino acids include, but are not limited to N- alpha-(9-Fluorenylmethyloxycarbonyl)-L-azidolysine (Fmoc-L-Lys(N3)-OH), N-alpha-(9- Fluorenylmethyloxycarbonyl)-L-biphenylalanine (Fmoc-L-Bip-OH), and N-alpha-(9- Fluorenylmethyloxycarbonyl)-O-benzyl-L-tyrosine (Fmoc-L-Tyr(Bzl)-OH, or their unprotected analogs.

[0212] Additionally, polymers may be added to IL-2 polypeptides. In some embodiments, the polymers are added in order to increase the half-life of the polypeptides. Such half-life extending polymers can be added to the N-terminus of the IL-2 polypeptides. The half-life extending polymers may be of any size, including up to about 6 kDa, up to about 30 kDa, or up to about 50 kDa. In some embodiments, the half-life extending polymers are PEG polymers.

[0213] In some embodiments, the IL-2 polypeptide comprises one or more amino acid substitutions or deletions selected from the Table 8 below, wherein residue numbering is based on SEQ ID NO: 701 as a reference sequence.Table 8 - Exemplary IL-2 SubstitutionsDocket No. 56146-746.601

[0214] In some embodiments, the IL-2 polypeptide comprises one or more amino acid substitutions selected from the Table 9 below, wherein residue numbering is based on SEQ IDNO: 701 as a reference sequence.Table 9 - Additional IL-2 SubstitutionsDocket No. 56146-746.601

[0215] The IL-2 polypeptides described herein may also be synthesized chemically rather than expressed as recombinant polypeptides. The IL-2 polypeptides can be made by synthesizing one or more fragments of the full-length IL-2 polypeptides, ligating the fragments together, and folding the ligated full-length polypeptide. In some embodiments, the IL-2 polypeptide comprises Y31H, K35R, Q74P, and C125S substitutions and optionally a PEG polymer covalently attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises Y31H, K35R, Q74P, N88D, and C125S substitutions and optionally a PEG polymer covalently attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the PEG polymer attached to the N-terminus acts as a linker which forms the attachment to the rest of the immunocytokine composition.

[0216] In some embodiments, the IL-2 polypeptides enhance regulatory T-cell (Treg) cell proliferation or activation when administered to a subject. In some embodiments, the IL-2 polypeptides enhance Treg proliferation or activation while sparing T-effector cells (Teff) and / or natural killer (NK) cells when administered to a subject. In some embodiments, the IL-2 polypeptides increase Treg cells without substantially increasing CD8+ T cells and NK cells when administered to a subject.

[0217] In some embodiments, an IL-2 polypeptide is biased in favor of activation of Treg cells compared to Teff cells. In some embodiments, the IL-2 polypeptide comprises at least one amino acid substitutions at residues selected from Y31, K35, Q74, and N88, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide comprises amino acid substitutions at each of residues Y31, K35, and Q74, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide comprises the amino acid substitutions of Y31H, K35R, and Q74P. In some embodiments, the IL-2 polypeptide comprises amino acid substitutions at each of residues Y31, K35, Q74, and N88, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide comprises the amino acid substitutions of Y31H, K35R, Q74P, and N88D. In some embodiments, the IL-2 polypeptide does not comprise any additional substitutions that have a substantial impact on the binding of the IL-2 polypeptide to the IL-2Ra receptor.Docket No. 56146-746.601

[0218] In some embodiments, the IL-2 polypeptide exhibits substantially lower ability to activate Teff cells than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide retains the ability to activate Treg cells. In some embodiments, the IL-2 polypeptide exhibits an enhanced ability to activate Treg cells compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702 In some embodiments, the IL-2 polypeptide exhibits at least about 4x lower dissociation constant (Kd) of IL-2Ra than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL- 2 polypeptide exhibits a 2-fold to 10-fold lower dissociation constant (Kd) of IL-2Ra than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702.

[0219] In some embodiments, an IL-2 polypeptide that exhibits a greater affinity for IL-2 receptor a subunit than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the affinity to IL-2 receptor a subunit is measured by dissociation constant (Kd). As used herein, the phrase “the Kd of the IL-2 polypeptide / IL-2 receptor a subunit” means the dissociation constant of the binding interaction of the IL-2 polypeptide and CD25.

[0220] In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit is less than 10 nM. In some embodiments the Kd of the IL-2 polypeptide / IL-2 receptor a subunit is less than 10 nM, less than 7.5 nM, less than 5 nM, less than 4 nM, or less than 3 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 1 nM and 0.1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 10 nM and about 0.1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 10 nM and about 1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 7.5 nM and about 0.1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 7.5 nM and about 1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 5 nM and about 0.1 nM. In some embodiments, the Kd of the IL-2 polypeptide / IL-2 receptor a subunit between about 5 nM and about 1 nM. In some embodiments, the Kd is measured by surface plasmon resonance.

[0221] In some embodiments, the IL-2 polypeptide that exhibits at least about a 10%, 50%, 100%, 250%, or 500% greater affinity for IL-2 receptor a subunit than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide exhibits at most about a 500%, 750%, or 1000% greater affinity for IL-2 receptor a subunit than an IL- 2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702.Docket No. 56146-746.601

[0222] In some embodiments, the IL-2 polypeptide exhibits about 1.5-fold to about 10-fold greater affinity for IL-2 receptor a subunit than an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702.

[0223] In some embodiments, the IL-2 polypeptide exhibits substantially the same binding affinity for the IL-2Ra as compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide exhibits a Kd with IL-2Ra that is within about 2-fold, about 4-fold, about 6-fold, about 8-fold, or about 10-fold of the Kd between an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702 and IL-2Ra.

[0224] In some embodiments, the IL-2 polypeptide exhibits reduced affinity for the IL-2 receptor 0 subunit (IL-2R0) as compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide exhibits at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, or at least about 500-fold fold lower affinity for the IL-2R.0. In some embodiments, the IL-2 polypeptide exhibits at least about 100-fold lower affinity for IL-2R.0. In some embodiments, the IL-2 polypeptide exhibits substantially no affinity for IL-2R.0. In some embodiments, the affinity is measured as the dissociation constant Kd (e.g., a lower affinity correlating with a higher dissociation constant).

[0225] In some embodiments, the IL-2 polypeptide exhibits a binding affinity for IL-2R.0 which is at least 500 nM, at least 1000 nM, at least 5000 nM, at least 10000 nM, at least 50000 nM, or at least 100000 nM. In some embodiments, the IL-2 polypeptide exhibits substantially no binding affinity for IL-2R.0.

[0226] In some embodiments, the IL-2 polypeptide exhibits an affinity for IL-2Ra which is at least about 30-fold greater, at least about 50-fold grater, at least about 75-fold greater, at least about 100-fold greater, at least about 500-fold greater, or at least about 1000-fold greater than for IL-2R.0. In some embodiments, the IL-2 polypeptide exhibits an affinity for IL-2Ra which is at least about 100-fold greater than for IL-2R0. In some embodiments, the IL-2 polypeptide exhibits an affinity for IL-2Ra which is at least about 1000-fold greater than for IL-2R.0.

[0227] In some embodiments, an IL-2 polypeptide described herein is capable of expanding a regulatory T-cell (Treg) cell population. In some embodiments, an IL-2 polypeptide described herein spares expansion of effector T-cells (Teff).

[0228] In some embodiments, an IL-2 polypeptide has a half maximal effective concentration (ECso) for activation of Treg cells that at most moderately reduced compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, activation of Treg cells is measured by assessing change in STAT5 phosphorylation in a population of T cellsDocket No. 56146-746.601 when in contact with the IL-2 polypeptide. In some embodiments, a Treg cell is identified by being CD4+, CD25+ and FoxP3+. In some embodiments, a Treg cell is identified by also showing elevated expression of CD25 (CD25H1). In some embodiments, the IL-2 polypeptide has an EC50 for activation of Treg cells of at most about 100 nM, at most about 75 nM, at most about 50 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, or at most about 25 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells of at most about 50 nM, at most about 40 nM, at most about 35 nM, at most about 30 nM, or at most about 25 nM, at most about 20 nM, at most about 15 nM, at most about 10 nM, or at most about 5 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells of at most about 100 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Treg cells of at most about 50 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells of at most about 25 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells of from about 0.1 nM to about 100 nM, from about 1 nM to about 100 nM, from about 0.1 nM to about 50 nM, from about 1 nM to about 50 nM, from about 0.1 nM to about 25 nM, from about 1 nM to about 25 nM, from about 0.1 nM to about 10 nM, or from about 1 nM to about 10 nM.

[0229] In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 2-fold, at most 5-fold, at most 10-fold, at most 20-fold, at most 50-fold, at most 100- fold, at most 200-fold, at most 500-fold, or at most 1000-fold greater compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Treg cells that is at most 2-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 5-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 10-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Treg cells that is at most 50-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Treg cells that is at most 100-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 200- fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 500-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Tregcells that is at most 1000-fold greater.

[0230] In some embodiments, an IL-2 polypeptide has a half maximal effective concentration (EC50) for activation of Teff cells that is substantially greater compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the Teff cell is 1, 2, or 3 of a CD8 Teff cell (e.g., CD8+), a Naive CD8 cell (e.g., CD8+, CD45RAQ, or a CD4 Con cellDocket No. 56146-746.601(e.g., CD4+, FoxP3‘), or any combination thereof. In some embodiments, activation of cells is measured by assessing change in STAT5 phosphorylation in a population of T cells when in contact with the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least about 10 nM, at least about 50 nM, at least about 100 nM, at least about 500 nM, at least about 1000 nM, at least about 2000 nM, at least about 3000 nM, at least about 4000 nM, or at least about 5000 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least about 100 nM. In some embodiments, the IL- 2 polypeptide has an EC50 for activation of Teff cells of at least about 500 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least about 1000 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least about 5000 nM. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold greater compared to an IL-2 polypeptide of SEQ ID NO: 701 and / or SEQ ID NO: 702. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 10-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 50-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 100-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 500-fold greater. In some embodiments, the IL-2 polypeptide has an EC50 for activation of Teff cells of at least 1000-fold greater.

[0231] In some embodiments, the IL-2 polypeptide exhibits a substantially greater ability to activate Treg cells compared to Teff cells. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Tregcell type is at least 10, at least 20, at least 50, at least 100, at least 150, or at least 200. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Tregcell type is at least 100. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Treg cell type is at least 200. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Tregcell type is at least 300. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Tregcell type is at least 500. In some embodiments, a ratio of EC50 for activation of a Teff cell type over EC50 for activation of a Tregcell type is at least 1000.

[0232] In some embodiments, the level of activation is measured after about 0.5 h to about Ih after incubation with the IL-2 polypeptide (e.g., 0.5 h to Ih before fixing the cells for in in vitro experiment).Docket No. 56146-746.601

[0233] In some embodiments, the IL-2 polypeptide comprising one or more amino acid substitutions. In some embodiments, the amino acid substitutions affect the binding properties of the IL-2 polypeptide to IL-2 receptor subunits (e.g. alpha, beta, or gamma subunits) or to IL-2 receptor complexes (e.g. IL-2 receptor aPy complex or J3y complex). In some embodiments, the amino acid substitutions are at positions on the interface of binding interactions between the IL-2 polypeptide and an IL-2 receptor subunit or an IL-2 receptor complex. In some embodiments, the amino acid substitutions cause an increase in affinity for the IL-2 receptor a|3y complex or alpha subunit. In some embodiments, the amino acid substitutions cause a decrease in affinity for the IL-2 receptor Py complex or beta subunit.

[0234] In some embodiments, the IL-2 polypeptide comprises natural amino acid substitutions relative to WT IL-2 (SEQ ID NO: 701). In some embodiments, the IL-2 polypeptide comprises up to seven natural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises up to six amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises up to five amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises up to four amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises up to three amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises from three to seven, three to six, three to five, three to four, four to seven, four to six, four to five, five to seven, five to six, or six to seven natural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises at least one, at least two, at least three, at least four, at least five, or at least six amino acid substitutions.

[0235] In some embodiments, an IL-2 polypeptide provided herein comprises natural amino acid substitutions at at least one of Y31, K35, Q74, and N88D wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide comprises natural amino acid substitutions at at least two of Y31, K35, Q74, and N88. In some embodiments, the IL-2 polypeptide comprises natural amino acid substitutions at at least three of Y31, K35, Q74, and N88. In some embodiments, the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises natural amino acid substitutions at each of Y31, K35, Q74, and N88. In some embodiments, the IL-2 polypeptide comprises the amino acid substitutions Y31H, K35R, Q74P, and N88D. In some embodiments, the IL-2 polypeptide further comprises an optional C125 substitution e.g., C125S or C125A). In some embodiments, the IL-2 polypeptide further comprises an optional Al deletion or substitution of residue AL In some embodiments, the IL-2 polypeptide further comprises an optional Al deletion.Docket No. 56146-746.601

[0236] In some embodiments, an IL-2 polypeptide provided herein comprises a Y31 substitution wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the Y31 substitution is for an aromatic amino acid. In some embodiments, the Y31 substitution is for a basic amino acid. In some embodiments, the basic amino acid is weakly basic. In some embodiments, the Y31 substitution is selected from Y31F, Y31H, Y31W, Y31R, and Y31K. In some embodiments, the Y31 substitution is Y31H.

[0237] In some embodiments, an IL-2 polypeptide provided herein comprises a K35 substitution, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the K35 substitution is for a basic amino acid. In some embodiments, the K35 substitution is for a positively charged amino acid. In some embodiments, the K35 substitution is K35R, K35E, K35D, or K35Q. In some embodiments, the K35 substitution is K35R.

[0238] In some embodiments, an IL-2 polypeptide provided herein comprises a Q74 substitution, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the Q74 substitution is a cyclic amino acid. In some embodiments, the cyclic amino acid comprises a cyclic group covalently attached to the alpha carbon and the nitrogen attached to the alpha carbon. In some embodiments, the Q74 substitution is Q74P.

[0239] In some embodiments, an IL-2 polypeptide provided herein comprises a N88 substitution, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the N88 substitution is a charged amino acid residue. In some embodiments, the N88 substitution is a negatively charged amino acid residue. In some embodiments, the N88 substitution is N88D or N88E. In some embodiments, the N88 substitution is N88D or N88E. In some embodiments, the N88 substitution is N88D.

[0240] In some embodiments, an IL-2 polypeptide comprises a C125 substitution, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the C125 substitution stabilizes the IL-2 polypeptide. In some embodiments, the C125 substitution does not substantially alter the activity of the IL-2 polypeptide. In some embodiments, the IL-2 polypeptide comprises a C125S substitution. In some embodiments, the IL-2 polypeptide comprises a C125A substitution.Docket No. 56146-746.601

[0241] In some embodiment, an IL-2 polypeptide comprises a modification at residue Al, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the modification is an Al deletion.

[0242] In some embodiments, the IL-2 polypeptide comprises additional amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises an additional amino acid substitution that has an effect on binding to the IL-2 receptor alpha subunit or aPy complex. In some embodiments, the IL-2 polypeptide comprises an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or Py complex. In some embodiments, the IL-2 polypeptide comprises at least one additional amino acid substitution selected from Table 8 or Table 9. In some embodiments, the IL-2 polypeptide comprises at least one amino acid substitution at residue E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the IL-2 polypeptide comprises at least one amino acid substitution at residue E15, N29, N30, T37, K48, V69, N71, 189, or 192. In some embodiments, the IL-2 polypeptide comprises 1, 2, 3, or 4 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. n some embodiments, the IL-2 polypeptide comprises 1, 2, 3, or 4 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, 189, or 192. In some embodiments, the IL-2 polypeptide comprises 1 natural amino acid substitutions at residues selected from El 5, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the IL-2 polypeptide comprises 2 In some embodiments, the IL-2 polypeptide comprises up to 2 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the IL-2 polypeptide comprises up to 3 natural amino acid substitutions at residues selected from E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the additional amino acid substitution comprises E15A, E15G, or E15S. In some embodiments, the additional amino acid substitution comprises N29S. In some embodiments, the additional amino acid substitution comprises N30S. In some embodiments, the additional amino acid substitution comprises T37A or T37R. In some embodiments, the additional amino acid substitution comprises K48E. In some embodiments, the additional amino acid substitution comprises V69A. In some embodiments, the additional amino acid substitution comprises N71R. In some embodiments, the additional amino acid substitution comprises N88A, N88D, N88E, N88F, N88G, N88H, N88I, N88M, N88Q, N88R, N88S, N88T, N88V, or N88W. In some embodiments, the additional amino acid substitution comprises N88D. InDocket No. 56146-746.601 some embodiments, the additional amino acid substitution comprises I89V. In some embodiments, the additional amino acid substitution comprises I92K or I92R.

[0243] In some embodiments, an IL-2 polypeptide provided herein comprises substitutions at Y31, K35, Q74, and optionally C125S. In some embodiments, the IL-2 polypeptide does not comprise any additional substitutions which substantially affect binding to the IL-2 receptor alpha subunit or aPy complex. In some embodiments, the IL-2 polypeptide does not comprise an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or Py complex. In some embodiments, the IL-2 polypeptide does not comprise any additional natural amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the IL-2 polypeptide does not comprise any additional amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, N88, 189, or 192. In some embodiments, the IL-2 polypeptide does not have a V69 substitution. In some embodiments, the IL-2 polypeptide does not have a V69A substitution. In some embodiments, the IL-2 polypeptide does not have a K48 substitution. In some embodiments, the IL-2 polypeptide does not have a K48E substitution. In some embodiments, the IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the IL-2 polypeptide does not comprise a substitution at either of V69 or K48. In some embodiments, the IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the IL-2 polypeptide does not comprise either a V69A or K48E substitution.

[0244] In some embodiments, an IL-2 polypeptide provided herein comprises substitutions at Y31, K35, Q74, N88, and optionally C125S. In some embodiments, the IL-2 polypeptide does not comprise any additional substitutions which substantially affect binding to the IL-2 receptor alpha subunit or aPy complex. In some embodiments, the IL-2 polypeptide does not comprise an additional amino acid substitution that has an effect on binding to the IL-2 receptor beta subunit or J3y complex. In some embodiments, the IL-2 polypeptide does not comprise any additional natural amino acid substitutions selected from positions identified in Table 8 or Table 9. In some embodiments, the IL-2 polypeptide does not comprise any additional amino acid substitutions selected from Table 8 or Table 9. In some embodiments, the IL-2 polypeptide does not comprise any additional natural amino acid substitutions at residues El 5, N29, N30, T37, K48, V69, N71, 189, or 192. In some embodiments, the IL-2 polypeptide does not comprise any additional amino acid substitutions at residues E15, N29, N30, T37, K48, V69, N71, 189, or 192. In some embodiments, the IL-2 polypeptide does not have a V69 substitution. In some embodiments, the IL-2 polypeptide does not have a V69A substitution. In someDocket No. 56146-746.601 embodiments, the IL-2 polypeptide does not have a K48 substitution. In some embodiments, the IL-2 polypeptide does not have a K48E substitution. In some embodiments, the IL-2 polypeptide does not comprise a substitution at V69 or K48. In some embodiments, the IL-2 polypeptide does not comprise a substitution at either of V69 or K48. In some embodiments, the IL-2 polypeptide does not comprise a V69A or K48E substitution. In some embodiments, the IL-2 polypeptide does not comprise either a V69A or K48E substitution.

[0245] In some embodiments, an IL-2 polypeptide provided herein comprises an N-terminal deletion. In some embodiments, the N-terminal deletion is of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acids. In some embodiments, the N-terminal deletion is of at least 1 amino acid. In some embodiments, the N-terminal deletion is of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the N-terminal deletion is from 1 to 15 amino acids. In some embodiments, the N-terminal deletion is a deletion of a single amino acid (e.g., an Al deletion of SEQ ID NO: 701).

[0246] n some embodiments, the unnatural amino acid substitutions provided herein can be incorporated into an IL-2 polypeptide in addition to any combination of natural amino acid substitutions provided herein, unless otherwise specified. For example, where an IL-2 polypeptide comprises, for example, Y31H, K35R, and Q74P natural amino acid substitutions is described, it is expressly contemplated that the IL-2 polypeptide can also comprise unnatural amino acid substitutions (e.g., Hse41, Hse71, Hsel04, Nle23, Nle39, and Nle46). As another example, where an IL-2 polypeptide provided herein is described as having Y31H, K35R, Q74P, and N88D natural amino acid substitutions, the IL-2 polypeptide can further comprise unnatural amino acid substitutions (e.g., Hse41, Hse71, Hsel04, Nle23, Nle39, and Nle46). In particular, any combination of natural amino acid substitutions present in a recombinant IL-2 polypeptide provided herein can also be incorporated into a synthetic version of the IL-2 polypeptide (e.g., the corresponding IL-2 polypeptide containing, for example, Hse41, Hse71, Hsel04, Nle23, Nle39, and Nle46).

[0247] In some embodiments, the IL-2 polypeptide comprises one or more unnatural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises at least two unnatural amino acid substitutions. In some embodiments, the IL-2 polypeptide comprises at least one amino acid substitution at a residue selected from Y31, K35, Q74, and N88, wherein residue position numbering of the IL-2 polypeptide is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the IL-2 polypeptide comprises a homoserine (Hse) residue located in any one of residues 36-45. In some embodiments, the IL-2 polypeptide comprises a Hse residue located in any one of residues 61-81. In some embodiments, the IL-2 polypeptideDocket No. 56146-746.601 comprises a Hse residue located in any one of residues 94-114. In some embodiments, the IL- 2 polypeptide comprises 1, 2, 3, or more Hse residues. In some embodiments, the IL-2 polypeptide comprises Hse41, Hse71, Hsel04, or a combination thereof. In some embodiments, the IL-2 polypeptide comprises Hse41, Hse71, and Hsel04. In some embodiments, the IL-2 polypeptide comprises at least two amino acid substitutions, wherein the at least two amino acid substitutions are selected from (a) a homoserine (Hse) residue located in any one of residues 36-45; (b) a homoserine residue located in any one of residues 61-81; and (c) a homoserine residue located in any one of residues 94-114. In some embodiments, the IL-2 polypeptide comprises Hse41 and Hse71. In some embodiments, the IL-2 polypeptide comprises Hse41 and Hsel04. In some embodiments, the IL-2 polypeptide comprises Hse71 and Hsel04. In some embodiments, the IL-2 polypeptide comprises Hse41. In some embodiments, the IL-2 polypeptide comprises Hse71. In some embodiments, the IL-2 polypeptide comprises Hsel04. In some embodiments, the IL-2 polypeptide comprises 1, 2, 3, or more norleucine (Nle) residues. In some embodiments, the IL-2 polypeptide comprises a Nle residue located in any one of residues 18-28. In some embodiments, the IL-2 polypeptide comprises one or more Nle residues located in any one of residues 34-50. In some embodiments, the IL-2 polypeptide comprises a Nle residue located in any one of residues 20- 60. In some embodiments, the IL-2 polypeptide comprises three Nle substitutions. In some embodiments, the IL-2 polypeptide comprises Nle23, Nle39, andNle46. In some embodiments, the IL-2 polypeptide comprises SEQ ID NO: 775. In some embodiments, the IL-2 polypeptide comprises SEQ ID NO: 775 with an Al deletion.

[0248] In some embodiments, the IL-2 polypeptide comprises an amino acid sequence of SEQ ID NO: 776. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence at least 85% identical to the sequence of SEQ ID NO: 775. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 98% identical to the sequence of SEQ ID NO: 775, wherein each residue which is substituted in SEQ ID NO: 775 relative to SEQ ID NO: 1 is retained.

[0249] In some embodiments, an IL-2 polypeptide described herein comprises 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%, or at least about 99% sequence identity to SEQ ID NO: 775. In some embodiments, an IL-2 polypeptide described herein comprises 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%, or at least about 99% sequence identity to SEQ ID NO: 775. In some embodiments, the sequence identity is measured by protein-protein BLAST algorithm usingDocket No. 56146-746.601 parameters of Matrix BLOSUM62, Gap Costs Existence: 11, Extension: !, and Compositional Adjustments Conditional Compositional Score Matrix Adjustment.

[0250] In some embodiments, the IL-2 polypeptide comprises a modification of a terminal residue (e.g., the N-terminal residue or the C-terminal residue) which comprises a polymer. In some embodiments, the modification to the terminal residue comprises the attachment of a conjugation handle to the terminal residue of the IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the IL-2 polypeptide through the N-terminal amino group or the C-terminal carboxyl group of the IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the IL-2 polypeptide through the N-terminal amino group of the IL-2 polypeptide. In some embodiments, the conjugation handle is attached to the N-terminal amino group of the IL-2 polypeptide through a glutaryl-amino-PEG linker. In some embodiments, the conjugation handle is attached to the N-terminal amino group of the IL-2 polypeptide through an adduct having a structurewherien each n is independently an integer from 1-30 (e.g., 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), and wherein X is a conjugation handle (e.g., an azide or other conjugation handle provided herein, such as a DBCO group). In some embodiments, the IL-2 polypeptide will comprise the adduct above, but the conjugation handle X is replaced with a reaction product of the conjugation handle and a complementary conjugation handle (e.g., a 1,2,3 triazole) linking the IL-2 polypeptide to an additional moiety (e.g., a larger polymer or an additional polypeptide). In some embodiments, the N-terminal amino group of the IL-2 polypeptide comprises an adduct having a structure

[0251] In some embodiments, a herein described IL-2 polypeptide comprises one or more polymers covalently attached thereon. In some embodiments, the described IL-2 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polymers covalently attached to the IL-2 polypeptide. In some embodiments, the described IL-2 polypeptide comprises a polymer covalently attached to the N-terminus of the IL-2 polypeptide. The polymers provided herein may attached directly to a residue of the IL-2 polypeptide, may be attached through a smallDocket No. 56146-746.601 linking group e.g., attached through a reaction with a conjugation handle incorporated into the IL-2 polypeptide).

[0252] The polymer as provided herein can be attached at any desired residue of the IL-2 polypeptide. In some embodiments, it is preferable that the polymer be attached at a residue which does not impact binding of the IL-2 polypeptide with the IL-2 receptor or a specific IL- 2 receptor subunit (e.g., the IL-2 receptor alpha subunit). In some embodiments, the polymer is attached at or near the N-terminus of the IL-2 polypeptide. In some embodiments, the polymer is attached to the N-terminus of the IL-2 polypeptide. In some embodiments, the N- terminus is residue Al of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence. In some embodiments, the N-terminus is residue P2 of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence (e.g., the IL-2 polypeptide comprises a deletion of residue Al from the sequence). In some embodiments, the polymer is attached at a residue position which blocks or diminished binding of the IL-2 polypeptide with the IL-2 receptor beta subunit. Such residues positions are provided in U.S. Patent Publication Number 20200231644A1, which is hereby incorporated by reference as if set forth herein in its entirety, and include, for example, residue positions K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126.

[0253] Non-limiting examples of IL-2 polypeptides which retain binding to the IL-2 receptor alpha but are detuned with respect to the beta and gamma subunits include those described in US20230303649A1. In some embodiments, an IL-2 polypeptide of an immunocytokine composition described herein is one described in US20230303649A1 (e.g., one described in Table 5 therein). In some embodiments, an IL-2 polypeptide of an immunocytokine composition described herein is one shown in Table 10 below.Table 10 - Exemplary IL-2 PolypeptidesModifications Which Reduce Heparin BindingDocket No. 56146-746.601

[0254] In some embodiments, an IL-2 polypeptide described herein (e.g., any of the IL-2 polypeptides described herein) exhibits reduced binding to heparin. In some embodiments, reducing the binding of the IL-2 polypeptide to heparin results an IL-2 polypeptide (or an immunocytokine composition) which exhibits improved in vivo stability, biodistribution, and / or PK properties. In some embodiments, the IL-2 polypeptide comprises one or more modifications which reduce the binding of the IL-2 polypeptide to heparin. Any of the IL-2 polypeptides described herein can include such modifications (e.g., any of the IL-2 polypeptides described herein as having reduced binding to the IL-2 receptor alpha, any of the IL-2 polypeptides described herein as having reduced binding to the IL-2 receptor beta, or any of the IL-2 polypeptides described herein described as retaining binding to the IL-2 receptor alpha with reduced binding to theIL-2 receptors beta and / or gamma, or an activatable IL-2 polypeptide described herein).

[0255] In some embodiments, the IL-2 polypeptide of a multifunctional immunocytokine described herein comprises a modified B’C’ loop region of the IL-2 polypeptide. The B’C’ loop region refers to the amino acids which form the linkage between helixes B and C of IL-2 (e.g., human IL-2). The B’C’ loop region contains the amino acids positioned between amino acids 73 and 84 of wild type human IL-2 (SEQ ID NO: 701). In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide an insertion of an exogenous peptide into the B’C’ loop region. In some embodiments, the modified B’C’ loop region of the IL-2 polypeptide comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide and an insertion of an exogenous peptide into the B’C’ loop region.

[0256] In some embodiments, the modified B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acids 73 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region comprises a deletion of each amino acid 73 and 84 of the IL-2 polypeptide. In some embodiments, the modified B’C’ loop region comprises a deletion of each amino acid 73 and 84 of the IL-2 polypeptide and insertion of an exogenous peptide.

[0257] In some embodiments, the modified B’C’ loop region comprises insertion of an exogenous peptide. In some embodiments, the exogenous peptide comprises the sequence GDGSIN (SEQ ID NO: 700). In some embodiments, the exogenous peptide consists of the sequence GDGSIN (SEQ ID NO: 700). In some embodiments, the modified B’C’ loop regionDocket No. 56146-746.601 comprises a deletion of each amino acid between amino acids 73 and 84 of the IL-2 polypeptide and an insertion of an exogenous peptide having the sequence GDGSIN (SEQ ID NO: 700) (i.e., the amino acids between 73 and 84 of the IL-2 polypeptide are replaced with the sequence GDGSIN (SEQ ID NO: 700)). In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-73 of SEQ ID NO: 701 and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 701. In some embodiments, the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 783.

[0258] In some embodiments, the IL-2 polypeptide of a multifunctional immunocytokine described herein comprises a substitution at one or more amino acids selected from residue 32, residue 35, residue 38, residue 76, residue 81, or residue 83. In some embodiments, the IL-2 polypeptide comprises a K32S, K35E, R38A, K76A, R81S, or R83S substitution. In some embodiments, the IL-2 polypeptide comprises K32S. In some embodiments, the IL-2 polypeptide comprises K35E. In some embodiments, the IL-2 polypeptide comprises R38A. In some embodiments, the IL-2 polypeptide comprises K76A. In some embodiments, the IL-2 polypeptide comprises R81S. In some embodiments, the IL-2 polypeptide comprises R83S. In some embodiments, the IL-2 polypeptide comprises K32S, K35E, and R38A substitutions. In some embodiments, the IL-2 polypeptide comprises K76A and R81S substitutions. In some embodiments, the IL-2 polypeptide comprises K76A, R81S, and R81S substitutions.Activatable IL-2 Polypeptides

[0259] In some embodiments, an IL-2 polypeptide described herein used in an immunocytokine composition (e.g., any of the IL-2 polypeptides described herein, such as the alpha-competent, beta-gamma detuned IL-2 polypeptides or the beta-gamma competent, alphadetuned IL-2 polypeptides) is an activatable IL-2 polypeptide. In some embodiments, the activatable IL-2 polypeptide comprises a cleavable peptide, in particular a cleavable peptide, attached to a side chain of a residue of the IL-2 polypeptide which, upon cleavage, converts the activatable IL-2 polypeptide into an active form (e.g., the IL-2 related activity of the IL-2 polypeptide enhances following cleavage).

[0260] Examples of activatable IL-2 polypeptides and strategies for generating activatable IL- 2 polypeptides compatible with the instant disclosure are described in WO2024150175A1 (corresponding US Patent Publication No. US20240417436A1), the contents of which are incorporated herein by reference as if set forth herein in its entirety. Further examples of suchDocket No. 56146-746.601 activatable IL-2 polypeptides incorporated into immunocytokines are described in WO2024150174A1 (corresponding US Patent Publication No. US20240417436A1), which is also incorporated herein by reference. Any of the strategies for generating an activatable IL-2 polypeptide described therein are contemplated as being applicable to any of the IL-2 polypeptides of the instant disclosure. In some embodiments, the activatable IL-2 polypeptide is one described in US Patent Publication No. US20240417436A1, in particularly in Table 3 therein (e.g., CMP-319 described therein).

[0261] Exemplary cleavable peptide sequences which can be incorporated into an activatable IL-2 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, US2021 / 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 (PCT) Publication Nos: WO2021 / 202675, WO2021 / 062406, WO2021 / 142471, WO202 1 / 216468, WO2021 / 119516, WO2021 / 253360, WO2021 / 146455, WO2021 / 202678, WO2021 / 202673, WO2021 / 189139, W02020 / 232303, WO2022 / 115865, and / or Chen et. al., J Bio Chem, 277, V6 P4485-4491. (2002), each of which is hereby incorporated by reference as if set forth herein in its entirety. In some embodiments, the cleavable peptide is one described in WO2024150172A1, the contents of which are incorporated by reference as if set forth herein in its entirety.Masked IL-2 Polypeptides and anti-IL-2 Masks

[0262] In some embodiments, an IL-2 polypeptide of an immunocytokine composition (i.e., any one of the IL-2 polypeptides described herein) is masked in the immunocytokine composition by a masking polypeptide. In some embodiments, the masking polypeptide is one which specifically binds to the IL-2 polypeptide, thereby disrupting the ability of the IL-2 polypeptide to bind with its receptor.

[0263] In some embodiments, the masking polypeptide is selected from an anti-IL-2 antigen binding fragment, an IL-2 receptor subunit polypeptide, or a steric blocking group. In some embodiments, the masing polypeptide is an anti-IL-2 antigen binding fragment (or another anti- IL-2 binding domain, such as any of the binding domain formats described herein). In some embodiments, the masking polypeptide is an anti-IL-2 VHH or scFv. In some embodiments, the masking polypeptide is an anti-IL-2 scFv. In some embodiments, the masking polypeptide is an IL-2 receptor subunit polypeptide. In some embodiments, the IL-2 receptor subunitDocket No. 56146-746.601 polypeptide is a binding sequence of an IL-2 receptor alpha subunit, and IL-2 receptor beta subunit, or an IL-2 receptor gamma subunit which is capable of binding to the IL-2 polypeptide (e.g., it acts as a “dummy receptor” of the IL-2 polypeptide). In some embodiments, the masking polypeptide is a steric blocking group (i.e., a bulky polypeptide that does not specifically interact with the IL-2 polypeptide but, by nature of its size, prevents or reduces binding of the IL-2 polypeptide with its receptor). In preferred embodiments, the masking polypeptide is an anti-IL-2 scFv.

[0264] In some preferred embodiments, the masking polypeptide (e.g., the anti-IL-2 scFv) is linked to the immunocytokine composition by a linker comprising a cleavable peptide (e.g., any of the cleavable peptide described herein, such as any of the protease cleavable peptide described herein). In some embodiments, the cleavable peptide is a protease cleavable peptide. In some embodiments, the cleavable peptide is cleavable by one or more proteases. In some embodiments, the cleavable peptide is cleavable by one or more proteases associated with a tumor or tumor microenvironment. In some embodiments, the cleavable peptide is preferentially or selectively cleaved in or near a tumor microenvironment. In some embodiments, the cleavable peptide is preferentially or selectively cleaved by one or more proteases associated with a tumor or tumor microenvironment.

[0265] 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 cleavable peptide is cleavable by an MMP. In some embodiments, the cleavable peptide is cleavable by a matriptase. In some embodiments, the cleavable peptide is cleavable by a plasminogen activator. In some embodiments, the cleavable peptide is cleavable by a legumain. In some embodiments, the cleavable peptide is cleavable by a protease set forth in Table 6 A.

[0266] In some embodiments, the cleavable peptide is cleavable by multiple proteases. In some embodiments, the cleavable peptide is cleavable by multiple classes of proteases. In some embodiments, the cleavable peptide is cleavable by 2, 3, or 4 different proteases. In some embodiments, the cleavable peptide comprises multiple cleavage sites. In some embodiments, the cleavable peptide comprises 2, 3, 4, or more cleavage sites. In some embodiments, the cleavable peptide comprises 2 cleavage sites. In some embodiments, the cleavable peptideDocket No. 56146-746.601 comprises 3 cleavage sites. In some embodiments, the cleavable peptide comprises 4 cleavage sites. In some embodiments, each of the cleavage sites is cleavable by a different protease.

[0267] In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease and a legumain. In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease and a matriptase. In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease and a plasminogen activator. In some embodiments, the cleavable peptide is cleavable by a legumain and a matriptase. In some embodiments, the cleavable peptide is cleavable by a legumain and a plasminogen activator. In some embodiments, the cleavable peptide is cleavable by a matriptase and a plasminogen activator. In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease, a legumain, and a matriptase. In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease, a matriptase, and a plasminogen activator. In some embodiments, the cleavable peptide is cleavable by a matrix metalloprotease, a legumain, and a plasminogen activator.

[0268] Exemplary cleavable peptide sequences which can be incorporated into an activatable IL-2 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, US2021 / 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 (PCT) Publication Nos: WO2021 / 202675, WO2021 / 062406, WO2021 / 142471, WO202 1 / 216468, WO2021 / 119516, WO2021 / 253360, WO2021 / 146455, WO2021 / 202678, WO2021 / 202673, WO2021 / 189139, W02020 / 232303, WO2022 / 115865, WO2021 / 202678, and / or Chen et. al., J Bio Chem, 277, V6 P4485-4491. (2002), each of which is hereby incorporated by reference as if set forth herein in its entirety. In some embodiments, the cleavable peptide is one described in WO2024150172A1, the contents of which are incorporated by reference as if set forth herein in its entirety.

[0269] In some embodiments, cleavage of the cleavable peptide releases the masking polypeptide from the immunocytokine composition, thereby dissociating the masking polypeptide from the immunocytokine composition and allowing the IL-2 polypeptide to exhibit its activity and / or bind to its receptor.

[0270] In some embodiments, the masking polypeptide (e.g., the anti-IL-2 scFv) is attached to the IL-2 polypeptide (e.g., it is fused to the N- or C-terminus of the IL-2 polypeptide via aDocket No. 56146-746.601 linker which comprises a cleavable peptide). Examples of such immunocytokine compositions are shown in FIGs. 7A-7I. In some embodiments, the masking polypeptide (e.g., the anti-IL-2 scFv) is attached to the immunocytokine composition at a different location. In some embodiments, the masking polypeptide is attached to the immunocytokine composition at a different chain of the Fc domain from which the IL-2 polypeptide is attached.Conjugation Vs. Fusion of IL-2 Polypeptide in Immunocytokine Compositions

[0271] In some embodiments, an IL-2 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-2 polypeptide as described herein is fused to another portion of the immunocytokine composition. In general, it is preferable to conjugate chemically synthesized IL-2 polypeptides as described herein to form the covalent attachment (e.g., by using AJICAP technology to suitable derivatize an Fc domain), whereas for IL-2 polypeptides described herein which can be recombinantly produced, it may be preferable to incorporate them as a fusion protein (e.g., by fusing the IL-2 polypeptide to a polypeptide chain of an Fc domain, such as shown in FIGs. 6A-6C, 7A-7G, and 8A-8H). In some embodiments, the IL-2 polypeptide is fused via its C-terminus, optionally via a peptide linker. In some embodiments, the 11-2 polypeptide is fused via its N-terminus, optionally via a peptide linker.

[0272] Non-limiting examples of IL-2 polypeptides of the instant disclosure which can be linked into an immunocytokine composition described herein via conjugation include those provided in the table below.Table 11 - Sequences of Exemplary Conjugatable IL-2 Polypeptides.Docket No. 56146-746.601Docket No. 56146-746.601

[0273] In the table above Nle refers to a norleucine residue, Hse refers to a homoserine residue, Phe(4COOH) refers to a 4-carboxyphenylalanine residue, Dab refers to 2,4-diaminobutyric acid residue, Om refers to an ornithine residue, and Tyr(Bzl) an O-benzyl-tyrosine residue.

[0274] In the table above, the listed SEQ ID NOs refer to the base sequences depicted in the table, and the “IL-2 Payload Name” refers to the conjugatable IL-2 polypeptide based on the corresponding SEQ ID NO. The structure of the conjugatable IL-2 polypeptide 2P72 is described supra. For each of conjugatable IL-2 polypeptides 2P73-2P80, the IL-2 polypeptide comprises an N-terminal modification of the structure:to provide a conjugation handle to the IL-2 polypeptide having the sequence noted in the SEQ ID NO listed in the table.Scaffolds for Multifunctional Immunocytokine Compositions

[0275] 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-2 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 ScaffoldsDocket No. 56146-746.601

[0276] 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-2 polypeptide).

[0277] In some embodiments, the Fc domain is conjugated to the cytokine (e.g., the IL-2 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 a 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.

[0278] 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.

[0279] 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%,Docket No. 56146-746.60190%, 95%, 96%, 97%, 98%, or 99% sequence identity with a corresponding natural Fc domain (e.g., each arm has the indicated sequence identity).

[0280] 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 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. 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 12 - Exemplary Fc SequencesDocket No. 56146-746.601

[0281] 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% sequenceDocket No. 56146-746.601 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 coextensive 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 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 13 - Exemplary Fc SequencesDocket No. 56146-746.601

[0282] 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.

[0283] 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 / fimmu.2017.00038) and include, forDocket No. 56146-746.601 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.

[0284] 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 group (such as a cytokine, in particular an IL-2 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-2 polypeptide as described herein). For example, in instances where AJICAP™ technology is intended to be used to conjugate the cytokine (e.g., the IL-2 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 K248ADocket No. 56146-746.601 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-2 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).

[0285] 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 CH3 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

[0286] 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-2 polypeptide) to the Fc domain.Docket No. 56146-746.601

[0287] 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-2 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-2 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-2 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-2 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 14 - Exemplary Bispecific Constructs Targeting VEGF and PD-1Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601Docket No. 56146-746.601

[0288] 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 nonterminal 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.

[0289] 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 (c.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) CH3Docket No. 56146-746.601 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).

[0290] 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.

[0291] 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 chemical 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.

[0292] 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.

[0293] 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.Docket No. 56146-746.601

[0294] 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-2 polypeptide described herein.Linker Points of Attachment to IL-2 Polypeptides

[0295] In some embodiments, immunocytokine compositions comprising an IL-2 polypeptide provided herein comprise linkers, including chemical linkers, which link the IL-2 polypeptide to the rest of the immunocytokine composition (e.g., the Fc domain of the immunocytokine composition). When an IL-2 polypeptide is used in an immunocytokine composition, the point of attachment of the linker which links to the IL-2 polypeptide can be any residue as provided herein.

[0296] In some embodiments, the linker is attached to an amino acid residue of the IL-2 polypeptide. In some embodiments, the linker is attached to any amino acid residue of the IL- 2 polypeptide (e.g., at a position corresponding to any one of positions 1-133 of SEQ ID NO: 701). In some embodiments, the linker is attached at a non-terminal residue (e.g., a residue other than the C-terminal residue or N-terminal residue) of the IL-2 polypeptide (e.g., a residue at position corresponding to any one of positions 2-132 of SEQ ID NO: 701). In some embodiments, the linker is attached at a non-terminal residue of the IL-2 polypeptide, wherein the IL-2 polypeptide has been extended or truncated by one or more amino acids relative to SEQ ID NO: 701 (e.g., the linker is attached to a residue corresponding to residue 2 of SEQ ID NO: 701 and residue 1 of SEQ ID NO: 701 has been deleted). In some embodiments, the linker is attached to the N-terminal residue of the IL-2 polypeptide. In some embodiments, the linker is attached to the N-terminal amine of the IL-2 polypeptide. In some embodiments, the linker is attached to the C-terminal residue of the IL-2 polypeptide. In some embodiments, the linker is attached to the C-terminal carboxyl group of the IL-2 polypeptide.

[0297] In some embodiments, the linker is attached to the IL-2 polypeptide at a residue in a region comprising residues 2-132, 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-2 polypeptide at a residue in ta region comprising residues 30-75. In some embodiments, the linker is attached to the IL-2 polypeptide at a residue in a region comprising residues 35-55, residues 35-50, residues 35-45, residues 30-50, residues 40-45, residues 60-75, residues 60-70,Docket No. 56146-746.601 residues 65-70, or residues 2-5. In some embodiments, the linker is attached to the IL-2 polypeptide at a residue selected from residue 65, 66, 67, 68, 69, and 70. In some embodiments, the linker is attached to the IL-2 polypeptide at a residue selected from residue 40, 41, 42, 43, 44, and 45. In some embodiments, the linker is attached to the IL-2 polypeptide at residue 42 or 45. In some embodiments, the linker is attached to the IL-2 polypeptide at residue 42. In some embodiments, the linker is attached to the IL-2 polypeptide at residue 45.

[0298] In some embodiments, the linker is attached to the IL-2 polypeptide at a residue which disrupts binding of the IL-2 polypeptide with the IL-2 receptor alpha subunit (IL-2Ra). Examples of these residues include residues 3, 5, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 65, 67, 68, 69, 71, 72, 103, 104, 105, and 107, as described in, for example, PCT Pub. Nos. WO2019028419A1, W02020056066A1, WO2021140416A2, and WO2021216478A1 each of which is hereby incorporated by reference as if set forth in its entirety. In some embodiments, the linker is covalently attached at a residue selected from residues corresponding to residues 3, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 65, 67, 68, 69, 71, 72, 103, 104, 105, and 107 of SEQ ID NO: 701. In some embodiments, the linker is covalently attached at residue 1, 35, 37, 38, 41, 42, 43, 44, 45, 60, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, or 107 of the IL-2 polypeptide In some embodiments, the linker is covalently attached at residue 5. In some embodiments, the linker is covalently attached at residue 38. In some embodiments, the linker is covalently attached at residue 42. In some embodiments, the linker is covalently attached at residue 45. In some embodiments, the linker is covalently attached at residue 61. In some embodiments, the linker is covalently attached at residue 65. In some embodiments, the linker is covalently attached at residue 68.

[0299] In some embodiments, such as those where the IL-2 polypeptide is desired to signal primarily through the IL-2 receptor alpha subunit (e.g., the alpha / beta / gamma receptor complex), the linker can be attached to a residue which disrupts binding of the IL-2 receptor to the IL-2 receptor beta subunit or IL-2 receptor gamma subunit.

[0300] In some embodiments, it is desirable that the point of attachment of the linker have no or minimal impact on the ability of the IL-2 polypeptide to interact with any receptor. In such cases, the linker is desirably attached via the N-terminal residue of the IL-2 polypeptide.

[0301] 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 isDocket No. 56146-746.601 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.

[0302] 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 any naturally occurring amino acid, or any unnatural amino acid (e.g., an amino acid containing a desired reactive group, 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.

[0303] 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 modified IL-2 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. Non-limiting examples of amino acid residues comprising conjugation handles can be found, for example, in PCT Pub. Nos. WO2015054658A1, WO2014036492 Al, and WO2021133839A1 W02006069246A2, and W02007079130A2, each of which is incorporated by reference as if set forth in its entirety.

[0304] In some embodiments, the linker is covalently attached at residue 42. In some embodiments, the linker is covalently attached at residue F42E, F42D, F42Q, F42K, F42N, or F42Y. In some embodiments, the linker is covalently attached at residue F42Y. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 42.

[0305] In some embodiments, the linker is covalently attached at residue 45. In some embodiments, the linker is covalently attached at residue Y45, Y45E, Y45C, Y45D, Y45Q, Y45K, or Y45N. In some embodiments, the linker is covalently attached at residue Y45. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 45.

[0306] In some embodiments, the linker is covalently attached at residue 65. In some embodiments, the linker is covalently attached at residue P65C, P65D, P65Q, P65E, P65N,Docket No. 56146-746.601P65K, or P65Y. In some embodiments, the linker is covalently attached residue P65C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 65.

[0307] In some embodiments, the linker is covalently attached at residue 5. In some embodiments, the linker is covalently attached at residue S5C, S5D, S5Q, S5K, S5N, S5K, or S5Y. In some embodiments, the linker is covalently attached residue S5C. In some embodiments, the linker is covalently attached to an unnatural amino acid at residue 5.

[0308] 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 or an amino-acid-PEG-alkyne group. In some embodiments, the modified amino acid a is a glutamate, aspartate, lysine, cysteine, or tyrosine modified to incorporate an azide, alkyne, or other conjugation handle 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, the modified amino acid a has a structure selected from:Docket No. 56146-746.601wherein each n is independently an integer from 1-30 and each m is independently an integer from 2-10. In some embodiments, n is an integer from 1-20, 1-10, 2-30, 2-20, 2-10, 5-30, 5-20,Docket No. 56146-746.601 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. In some embodiments, each m is independently 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6. The modified amino acid a can be incorporated at any point of attachment of the IL-2 polypeptide as provided herein. In some embodiments, the modified amino acid a is located at a position on the IL-2 polypeptide selected from residue 5, residue 42, residue 45, or residue 65. In some embodiments, the modified amino acid a is located at residue 42 of the IL-2 polypeptide. In some embodiments, the modified amino acid a is located at residue 45 of the IL-2 polypeptide.

[0309] In some embodiments, the linker is attached to a terminal residue of the IL-2 polypeptide. In some embodiments, the linker is attached to the N-terminal residue of the IL-2 polypeptide. In some embodiments, the linker is attached to the N-terminal amine of the IL-2 polypeptide. In some embodiments, the linker is attached to the N-terminal amine of the IL-2 polypeptide through use of a conjugation handle attached to the N-terminal amine of the IL-2 polypeptide. In some embodiments, the linker is attached to the N-terminal amine of the IL-2 polypeptide through use of a conjugation handle attached to the N-terminal amine of the IL-2 polypeptide through a PEG group. In some embodiments, the conjugation handle comprises an azide or alkyne functionality. In some embodiments, the linker is attached to the N-terminal amine of the IL-2 polypeptide by use of a structurewherein each n is independently an integer from 1-30 and each m is independently an integer from 2-10. 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. In someDocket No. 56146-746.601 embodiments, each m is independently 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

[0310] In some embodiments, the linker is attached to the C-terminal residue of the IL-2 polypeptide. In some embodiments, the linker is attached to the C-terminal carboxyl group of the 11-2 polypeptide. In some embodiments, the linker is attached to the C-terminal carboxyl of the IL-2 polypeptide through use of a conjugation handle attached to the C-terminal carboxyl of the IL-2 polypeptide. In some embodiments, the linker is attached to the C-terminal carboxyl of the IL-2 polypeptide through use of a conjugation handle attached to C-terminal carboxyl of the IL-2 polypeptide through a PEG group. In some embodiments, the conjugation handle comprises an azide or alkyne functionality.

[0311] Where IL-2 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-2 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.Methods For Attaching Linkers to Fc Domains

[0312] 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-2 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

[0313] 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 orDocket No. 56146-746.601 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.

[0314] 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.

[0315] An exemplary affinity peptide with cleavable linker and conjugation handle payload capable of attaching the payload to residue K248 of Fc domain as provided herein can be found as reported 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.

[0316] 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 Second Generation: 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)).

[0317] 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 oxy vinyl group; a thiopropanoate group; an ethane- 1,2-diol group; an (imidazole- 1- 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 acetamidoethylDocket No. 56146-746.601 phosphoramidite 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.

[0318] In some embodiments, the cleavable linker is:Docket No. 56146-746.601wherein:-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.

[0319] 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-hydroxysuccinimide 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:Docket No. 56146-746.601, 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.

[0320] 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., alphahalogenated carbonyl group, alpha-beta unsaturated carbonyl group, mal eimide group, etc. .

[0321] 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 lysineof the Fc 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. In 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.

[0322] 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 linkingDocket No. 56146-746.601, p y g1-6 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-2 polypeptide (e.g., at any of the residues provided herein, such as C68)).

[0323] In some embodiments of immunocytokines described herein, the bifunctional linking reagent used has the structure

[0324] 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 is cleaved and the conjugation handle is immediately ready for subsequent conjugation to the cytokine (e.g., the IL-2 polypeptide).Alternative Methods of Modifying Fc RegionDocket No. 56146-746.601

[0325] 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.

[0326] In some embodiments, a cytokine (e.g., an IL-2 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 sitespecific 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-2 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-2 polypeptide) is later reacted with the conjugation handle to form the immunocytokine.

[0327] In some embodiments, a cytokine (e.g. IL-2 polypeptide) can be conjugated to a suitable Fc domain utilizing a transpeptide-mediated strategy for either direct attachment of the cytokine (e.g., IL-2 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.

[0328] In another aspect, the disclosure provides methods of generating immunocytokines using an engineered Fc-containing polypeptide conjugate comprising the formula: (Fc- containing polypeptide-T-A), wherein T is an acyl donor glutamine-containing tag engineeredDocket No. 56146-746.601 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 po...

Claims

Docket No. 56146-746.601CLAIMSWHAT 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-2 polypeptide.

4. The composition of claim 3, wherein the IL-2 polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to wild type IL-2 (SEQ ID NO: 701).

5. The composition of claim 3 or 4, wherein the IL-2 polypeptide has reduced affinity for the IL-2 receptor beta subunit compared to the IL-2 of SEQ ID NO: 701.

6. The composition of any one of claims 3-5, wherein the IL-2 polypeptide exhibits a reduced ability to signal through the IL-2 receptor beta / gamma complex compared to the IL-2 of SEQ ID NO: 701.

7. The composition of any one of claims 3-6, wherein the IL-2 polypeptide retains the ability to bind to the IL-2 receptor alpha subunit and exhibits a diminished ability to bind to the IL-2 receptor beta or gamma subunits relative to SEQ ID NO: 701.

8. The composition of any one of claims 3-7, wherein the IL-2 polypeptide has reduced affinity for heparin compared to the IL-2 of SEQ ID NO: 701.

9. The composition of any one of claims 3-8, wherein the IL-2 polypeptide comprises a modified B’C’ loop region, wherein the modified B’C’ loop region comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide and insertion of an exogenous peptide into the B’C’ loop region, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence.

10. The composition of claim 9, wherein the inserted peptide comprises the sequence GDGSIN (SE ID NO: 700).Docket No. 56146-746.60111. The composition of claim 10, wherein the inserted peptide consists of the sequence GDGSIN.

12. The composition of any one of claims 9-11, wherein the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acids 73 and 84 of the IL-2 polypeptide.

13. The composition of any one of claims 9-12, wherein the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of each of amino acids 74-83 of the IL-2 polypeptide.

14. The composition of any one of claims 3-13, wherein the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-73 of SEQ ID NO: 701 and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 501.

15. The composition of any one of claims 3-14, wherein the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 783.

16. The composition of any one of claims 3-15, wherein the IL-2 polypeptide comprises a K76A or R81S substitution, or both.

17. The composition of any one of claims 3-16, wherein the IL-2 polypeptide comprises polypeptide comprises an amino acid substitution at residue N88.

18. The composition of any one of claims 3-17, wherein the IL-2 polypeptide comprises an N88D substitution.

19. The composition of any one of claims 3-18, wherein the IL-2 polypeptide comprises a substitution at any one of residues L12, E15, L19, T123, Q126, or 1129.

20. The composition of any one of claims 3-19, wherein the IL-2 polypeptide comprises one or more substitutions selected from L12A, L12Y, E15D, E15S, L19A, L19D, T123A, Q126T, I129A, and I129K.

21. The composition of any one of claims 3-20, wherein the IL-2 polypeptide comprises any one of the following sets of substitutions:Q126T;I129K;I129A, E15S, T123A;E15D;L12A, L19A, E15S;Docket No. 56146-746.601L12Y, L19D;L12A, L19A; orL19D.

22. The composition of any one of claims 3-21, wherein the IL-2 polypeptide comprises an E15D or an L19D substitution.

23. The composition of any one of claims 3-22, wherein the IL-2 polypeptide comprises a T3A substitution.

24. The composition of any one of claims 3-23, wherein the IL-2 polypeptide comprises a C125S substitution.

25. The composition of any one of claims 3-8, comprising the sequence set forth in any one of SEQ ID NOs: 702-783.

26. The composition of any one of claims 3-25, wherein the IL-2 polypeptide is in covalent association via a fusion of the IL-2 polypeptide to the portion of the composition to which it is attached.

27. The composition of claim 26, wherein the IL-2 polypeptide is fused via its C-terminus.

28. The composition of any one of claims 1-27, 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).

29. The composition of any one of claims 1-28, wherein the first binding domain is an antigen binding fragment derived from an antibody.

30. The composition of any one of claims 1-29, 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).

31. The composition of claim 30, 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, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

32. The composition of claim 30 or 31, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 1A, IB, or 1C.

33. The composition of any one of claims 30-32, wherein the VH comprise an amino acid sequence of a VH set forth in Table 1 A , IB, or 1C.Docket No. 56146-746.60134. The composition of any one of claims 30-33, wherein the first binding domain is a VHH.

35. The composition of claim 34, 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; 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.

36. The composition of claim 34 or 35, 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.

37. The composition of any one of claims 30-33, 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).

38. The composition of any one of claims 30-33 or 37, 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.

39. The composition of any one of claims 1-33 or 37-38, 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 complementaryDocket No. 56146-746.601 determining region (VL CDR2), and a light chain third complementary determining region (VL CDR3).

40. The composition of claim 39, 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.

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

42. The composition of any one of claims 39-41, wherein 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 LASYLE (SEQ ID NO: 117), and a VL CDR3 sequence of QHSRELPLT (SEQ ID NO: 118).

43. The composition of any one of claims 39-42, 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.

44. The composition of any one of claims 1-33 or 37-43, 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 RDYRFDMGFD Y (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; orDocket No. 56146-746.601 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).

45. The composition of anyone of claims 1-33 or 37-44, 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.

46. The composition of any one of claims 1-33 or 37-45, wherein the first binding domain is an scFv.

47. The composition of any one of claims 1-33 or 37-46, wherein the first binding domain is a Fab.

48. The composition of any one of claims 1-47, wherein the second binding domain targeting VEGFA is capable of disrupting the interaction of VEGFA with one or more of its receptors.

49. The composition of any one of claims 1-48, wherein the second binding domain is comprised in an antigen binding fragment derived from an antibody.

50. The composition of any one of claims 1-49, 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).Docket No. 56146-746.60151. The composition of claim 50, 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, a bispecific diabody, triabody, a tetrabody, a maxibody, a camelid, a VHH, a Fab-Fc, a scFv-Fc, or a bispecific antibody.

52. The composition of claim 50 or 51, wherein the VH comprises a set of VH CDR1, VH CDR2, and VH CDR3 derived from an antibody in Table 2 A or 2B.

53. The composition of any one of claims 50-52, wherein the VH comprises an amino acid sequence of a VH set forth in Table 2A, 2B, or 2C.

54. The composition of any one of claims 50-53, 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).

55. The composition of any one of claims 50-54, 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.

56. The composition of any one of claims 1-55, 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).

57. The composition of claim 56, 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.

58. The composition of claim 56 or 57, wherein the VL comprises a set of VL CDR1, VL CDR2, and VL CDR3 derived from an antibody in Table 2 A or 2B.

59. The composition of any one of claims 56-58, 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).Docket No. 56146-746.60160. The composition of any one of claims 56-59, 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.

61. The composition of any one of claims 1-60, 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 a sequence FTSSLHS (SEQ ID NO: 129), and a VL CDR3 having a sequence QQYSTVPWT (SEQ ID NO: 130).

62. The composition of any one of claims 1-61, 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: 12763. The composition of any one of claims 1-62, wherein the second binding domain is an scFv.

64. The composition of any one of claims 1-63, wherein the second binding domain is a Fab.

65. The composition of any one of claims 1-53, wherein the second binding domain is a single domain antibody.

66. The composition of claim 65, wherein the second binding domain is a single domain heavy chain antibody (VHH).

67. The composition of claim 65 or 66, 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);Docket No. 56146-746.601 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 SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

68. The composition of any one of claims 65-67, 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.

69. The composition of claim 65, wherein the second binding domain comprises a light chain single domain antibody.

70. The composition of claim 65 or 69, 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).

71. The composition of any one of claims 65, 69, or 70, 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.

72. The composition of any one of claims 1-48, wherein the second binding domain is an anti-VEGFA anticalin.

73. The composition of any one of claims 1-48 or 72, 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.

74. The composition of any one of claims 1-73, wherein the composition comprises multiple copies of the first binding domain, the second binding domain, or both.

75. The composition of any one of claims 1-74, wherein the composition comprises multiple copies of the second binding domain.

76. The composition of claim 75, wherein the composition comprises two copies of the second binding domain.

77. The composition of claim 76, wherein one of the copies of the second binding domain contains an extension of one or more amino acids on the second binding domain relative to the other copy.Docket No. 56146-746.60178. The composition of any one of claims 74-77, wherein the multiple copies of the second binding domain are present on the same polypeptide chain.

79. The composition of any one of claims 74-77, wherein the multiple copies of the second binding domain are present on different polypeptide chains.

80. The composition of any one of claims 1-79, 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.

81. The composition of claim 80, wherein the Fc domain is derived from an IgG.

82. The composition of claim 81, wherein the Fc domain is derived from an IgGl or IgG4.

83. The composition of any one of claims 80-82, wherein the composition comprises a structure of the formula: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, a masking polypeptide for 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, a masking polypeptide for the cytokine, 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.Docket No. 56146-746.60184. The composition of claim 83, 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, the cytokine, 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.

85. The composition of claim 84, wherein X’ is the cytokine and C and C’ are both absent.

86. The composition of claim 84, wherein X’ is the copy of the first binding domain and one of C or C’ is the cytokine.

87. The composition of claim 84, wherein X’ is the copy of the first binding domain, one of Z or Z’ is the second binding domain and the other is the cytokine, and both C and C’ are absent.

88. The composition of any one of claims 84-86, 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.

89. The composition of claim 83, 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, the cytokine, 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.

90. The composition of claim 89, wherein X’ is the cytokine and C and C’ are both absent.

91. The composition of claim 89, wherein X’ is a copy of the second binding domain one of C or C’ is the cytokine.

92. The composition of claim 88, wherein X’ is the copy of the second binding domain, one of Z or Z’ is the second binding domain and the other is the cytokine, and both C and C’ are absent.

93. The composition of any one of claims 89-91, 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.

94. The composition of claim 83, 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.

95. The composition of claim 94, wherein:Z is the third binding domain and Z’ is absent;Z is absent and Z’ is the fourth binding domain; orDocket No. 56146-746.601 both Z and Z’ are absent.

96. The composition of claim 83, wherein X is the first binding domain, X’ is the second binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and both C and C’ are absent.

97. The composition of claim 83 or 96, wherein the masking polypeptide for the cytokine is attached to the Y or Y’ to which it is connected via a cleavable peptide linker.

98. The composition of any one of claims 83, 96, or 97, 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.

99. The composition of any one of claims 83, 96, or 97, 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.

100. The composition of any one of claims 83-99, wherein X is one of the first or second binding domains and is a Fab, VHH, or scFv.

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

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

103. The composition of any one of claims 100-102, 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.

104. The composition of claim 83, 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; orDocket No. 56146-746.601X 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; orX 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; orX is a Fab and is the first binding domain, X’ is Fab or ScFv and is the second binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and C and C’ are both absent; orX is a Fab and is the second binding domain, X’ is a Fab or scFv and is the first binding domain, one of Z or Z’ is the cytokine and the other is the masking polypeptide for the cytokine, and C and C’ are both absent.

105. The composition of any one of claims 3-79, 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.

106. The composition of claim 105, 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 RDYRFDMGFD Y (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); orDocket No. 56146-746.601 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).

107. The composition of claim 105 or 106, 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.

108. The composition of any one of claims 105-107, wherein the first polypeptide chain comprises, in N- to C-terminal direction, the VL and light chain constant region.

109. The composition of claim 108, 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.

110. The composition of any one of claims 105-109, wherein the first polypeptide chain comprises the sequenceEIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIY LASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKDocket No. 56146-746.601VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.H E The composition of any one of claims 105-110, 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.

112. The composition of claim 111, wherein the antibody constant region is an IgGl or IgG4 constant region.

113. The composition of claim 111 or 112, 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.

114. The composition of claim 113, wherein 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.

115. The composition of any one of claims 111-114, 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.

116. The composition of claim 115, 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).

117. The composition of claim 115 or 116, wherein the second binding domain is a VHH.

118. The composition of claim 117, wherein the VHH comprises a two proline peptide on its C-terminus.Docket No. 56146-746.601119. The composition of claim 117 or 118, 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); 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 SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

120. The composition of any one of claims 117-119, 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.

121. The composition of any one of claims 117-120, 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).

122. The composition of claim 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 SEQ ID NO:200, 217, or 221.

123. The composition of any one of claims 115-122, wherein the second polypeptide chain further comprises an additional binding domain targeting VEGFA.

124. The composition of any one of claims 115-123, wherein the additional binding domain targeting VEGFA is a VHH.Docket No. 56146-746.601125. The composition of claim 123 or 124, 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.

126. The composition of claim 124 or 125, 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 SRAYGSSRLRLADTYEY (SEQ ID NO: 220).

127. The composition of any one of claims 124-126, 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.

128. The composition of any one of claims 123-127, wherein 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.

129. The composition of claim 128, 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)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

130. The composition of any one of claims 105-129, wherein the second polypeptide chain comprises the sequence set forth in any one of SEQ ID NOs: 163, 164, 169, or 171.

131. The composition of any one of claims 105-130, wherein the third polypeptide chain comprises, in an N-terminal to C-terminal direction, the IL-2 polypeptide, an optional peptide linker, and an antibody constant region.

132. The composition of claim 131, 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 GGGGSDocket No. 56146-746.601(SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

133. The composition of claim 131 or 132, 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.

134. The composition of claim 133, 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).

135. The composition of claim 133 or 134, wherein the second binding domain or the additional binding domain targeting VEGFA is a VHH.

136. The composition of claim 135, wherein the VHH comprises a two proline peptide on its C-terminus.

137. The composition of claim 135 or 136, 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).

138. The composition of claim 137, 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.

139. The composition of any one of claims 131-138, wherein the third polypeptide comprises the second binding domain and the additional binding domain targeting VEGFA separated by an optional peptide linker.

140. The composition of claim 139, 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 GGGGSDocket No. 56146-746.601(SEQ ID NO: 21), (GGGGS)2(SEQ ID NO: 22), (GGGGS)3(SEQ ID NO: 29), or (GGGGS)4(SEQ ID NO: 30).

141. The composition of claim 139 or 140, 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.

142. The composition of any one of claims 131-141, wherein the antibody constant region is an IgGl or IgG4 constant region, or a portion thereof.

143. The composition of any one of claims 131-142, wherein the antibody constant region comprises, in an N-terminal to C-terminal direction, a hinge region, a CH2 domain, and a CH3 domain.

144. The composition of claim 142 or 143, 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.

145. The composition of claim 144, wherein 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.

146. The composition of any one of claims 131-145, wherein the third polypeptide chain comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 802, 803, 806, or 807.

147. The composition of claim 105, 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, or 171; and the third polypeptide chain comprises the amino acid sequence of any one of SEQ ID NOs: 802, 803, 806, or 807.Docket No. 56146-746.601148. The composition of claim 105, 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 802;SEQ ID NOs: 47, 163, and 803;SEQ ID NOs: 47, 164, and 806;SEQ ID NOs: 47, 164, and 807;SEQ ID NOs: 47, 169, and 802;SEQ ID NOs: 47, 169, and 803;SEQ ID NOs: 47, 172, and 802;SEQ ID NOs: 47, 172, and 803;SEQ ID NOs: 47, 163, and 806;SEQ ID NOs: 47, 163, and 807;SEQ ID NOs: 47, 169, and 806;SEQ ID NOs: 47, 169, and 807;SEQ ID NOs: 47, 171, and 809; orSEQ ID NOs: 47, 171, and 810.

149. The composition of any one of claims 3-79, wherein the second binding domain is a Fab having a VH and a VL, 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.

150. The composition of claim 149, wherein second binding domain Fab comprises a VH having a VH CD1, VH CDR2, and VH CDR3 and a VL having a VL CDR1, CDR2, and CDR3 of any one of the anti-VEGFA antibodies in Table 2A, optionally wherein the VH comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the corresponding VL.

151. The composition of claim 149 or 150, wherein the VH CDR1, VH CDR2, and VH CDR3 are SEQ ID NOs: 123, 124, and 125, respectively and the VL CDR1, VL CDR2, and VL CDR3 are SEQ ID NOs: 128, 129, and 130, respectively.

152. The composition of claim 151, wherein the VH and VL each have a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs: 122 and 126, respectively.Docket No. 56146-746.601153. The composition of any one of claims 149-152, wherein the first polypeptide chain comprises, in N- to C-terminal direction, the VL and a light chain constant region.

154. The composition of claim 153, 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, 277, or 278.

155. The composition of any one of claims 149-154, wherein the second polypeptide chain comprises, in an N-terminal to C-terminal direction, the VH of the Fab of the second binding domain and an antibody constant region.

156. The composition of claim 155, wherein the antibody constant region is an IgGl or an IgG4.

157. The composition of claim 155, 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.

158. The composition of claim 157, wherein 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, 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 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.

159. The composition of claim 157 or 158, 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 first binding domain specific for PD-1.

160. The composition of claim 159, wherein the optional peptide linker is present and comprises a sequence of any one of SEQ ID NOs: 21-30.

161. The composition of any one of claims 149-160, wherein the first binding domain is a VHH.

162. The composition of claim 161, wherein the VHH comprises a VH CDR1, VH CDR2, and VH CDR3 of any one of the anti-PD-1 VHH in Table IB or Table 1C.

163. The composition of claim 161, wherein the anti-PD-1 binding domain comprises the CDRS of VHH47, VHH62, VHH70, VHH76, VHH84, or VHH178.Docket No. 56146-746.601164. The composition of claim 163, wherein the first 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 VHH47, VHH62, VHH70, VHH76, VHH84, or VHH178.

165. The composition of claim 164, wherein the first binding domain is VHH47.

166. The composition of any one of claims 159-164, wherein the second polypeptide chain further comprises an additional binding domain targeting PD-1.

167. The composition of claim 166, wherein the additional binding domain VHH comprises an identical amino acid sequence compared to the first binding domain.

168. The composition of claim 166 or 167, wherein the first binding domain and the additional binding domain are separated by a peptide linker, optionally wherein the peptide linker comprises a sequence of any one of SEQ ID NOs: 22-30.

169. The composition of any one of claims 149-168, wherein the third polypeptide chain comprises, in an N-terminal to C-terminal direction, a VH of a second Fab specific for VEGFA, an antibody constant region, an optional peptide linker, and the IL-2 polypeptide.

170. The composition of claim 169, wherein the second Fab is the same as the Fab of the second binding domain.

171. The composition of claim 169 or 170, wherein the antibody constant region of the third polypeptide is an IgGl or an IgG4..

172. The composition of any one of claims 169-171, wherein the antibody constant region of the third polypeptide comprises, in an N-terminal to C-terminal direction, a CHI domain, a hinge region, a CH2 domain, and a CH3 domain.

173. The composition of claim 172, wherein 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, 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 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.

174. The composition of any one of claims 169-173, wherein the optional peptide linker of the third polypeptide is present and comprises a sequence of any one of SEQ ID NOs: 21-30.

175. The composition of any one of claims 149-174, wherein the IL-2 polypeptide comprises the sequence set forth in SEQ ID NO: 751, 753, 754, or 758.Docket No. 56146-746.601176. The composition of any one of claims 1-175, wherein the composition comprises only one cytokine.

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

178. A host cell comprising the composition of any one of claims 1-176 or the one or more polynucleotides of claim 77.

179. A pharmaceutical composition comprising the composition of any one of claims 1-150, and a pharmaceutically acceptable carrier or excipient.

180. 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 1-176 or the pharmaceutical composition of claim 179.

181. An IL-2 polypeptide comprising a modified B’C’ loop region, wherein the modified B’C’ loop region comprises a deletion of one or more amino acids of the B’C’ loop region between amino acids 73 and 84 of the IL-2 polypeptide, wherein residue position numbering is based on SEQ ID NO: 701 as a reference sequence, and an insertion of a peptide comprising the sequence GDGSIN into the deleted portion of the B’C’ loop region.

182. The IL-2 polypeptide of claim 181, wherein the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids between amino acids 73 and 84 of the IL-2 polypeptide.

183. The IL-2 polypeptide of claim 181 or 182, wherein the deletion of one or more amino acids of the B’C’ loop region comprises a deletion of each of amino acids 74- 83 of the IL-2 polypeptide.

184. The IL-2 polypeptide of any one of claims 181-183, wherein the inserted peptide consists of the sequence GDGSIN.

185. The IL-2 polypeptide of any one of claims 181-184, wherein the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 1-73 of SEQ ID NO: 701 and a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to amino acids 84-133 of SEQ ID NO: 701.

186. The IL-2 polypeptide of any one of claims 154-158, wherein the IL-2 polypeptide comprises a peptide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 783.Docket No. 56146-746.601187. An IL-2 polypeptide comprising the amino acid substitutions K76A and R81S, wherein residue position numbering is based on SEQ ID NO: 1 as a reference sequence.

188. The IL-2 polypeptide of any one of claims 181-187 claim 1, wherein the IL-2 polypeptide exhibits reduced binding to heparin compared to the IL-2 polypeptide of SEQ ID NO: 701.

189. The IL-2 polypeptide of any one of claims 181-188, wherein the IL-2 polypeptide exhibits reduced binding to the IL-2 receptor beta subunit compared to the IL-2 of SEQ ID NO: 1.

190. The IL-2 polypeptide of any one of claims 181-189, wherein the IL-2 polypeptide comprises an amino acid substitution at residue N88.

191. The IL-2 polypeptide of any one of claims 181-189, wherein the IL-2 polypeptide comprises an N88D substitution.

192. The IL-2 polypeptide of any one of claims 181-191, wherein the IL-2 polypeptide exhibits reduced binding to the IL-2 receptor gamma subunit.

193. The IL-2 polypeptide of any one of claims 181-192, wherein the IL-2 polypeptide comprises a substitution at any one of residues L12, E15, L19, T123, Q126, or 1129.

194. The IL-2 polypeptide of any one of claims 181-193, wherein the IL-2 polypeptide comprises one or more substitutions selected from L12A, L12Y, E15D, E15S, L19A, L19D, T123A, Q126T, or I129A, I129K.

195. The IL-2 polypeptide of any one of claims 181-194, wherein the IL-2 polypeptide comprises any one of the following sets of substitutions: Q126T;I129K;I129A, E15S, T123A;E15D;L12A, L19A, E15S;L12Y, L19D;L12A, L19A; or L19D.

196. The IL-2 polypeptide of any one of claims 181-195, wherein the IL-2 polypeptide comprises an E15D or an L19D substitution.Docket No. 56146-746.601197. The IL-2 polypeptide of any one of claims 181-196, comprising a T3A substitution.

198. The IL-2 polypeptide of any one of claims 181-197, comprising a C125S substitution.

199. The IL-2 polypeptide of any one of claims 181-198, wherein the IL-2 polypeptide binds to the IL-2 receptor alpha subunit.

200. The IL-2 polypeptide of any one of claims 181-199, comprising the sequence set forth in any one of SEQ ID NOs: 703-774.

201. A fusion polypeptide comprising the IL-2 polypeptide of any one of claims 181-200 and an additional polypeptide.

202. The fusion polypeptide of claim 201, wherein the IL-2 polypeptide is fused to the additional polypeptide at its C-terminus.

203. The fusion polypeptide of claim 201 or 202, wherein the additional polypeptide comprises an Fc domain.

204. The fusion polypeptide of claim 203, wherein the IL-2 polypeptide is connected to the Fc domain via a peptide linker.

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