EP4713356A1 - Pd-1-regulated il-2 immunocytokine and uses thereof - Google Patents

Abstract

The present invention provides PD-1 -regulated IL-2 immunoconjugates and compositions thereof. The invention also features polynucleotides, vectors, host cells, methods of production, pharmaceutical compositions, methods of treating a disease or disorder, such as cancer, related uses and compositions for use, and kits for use with the one or more methods.

Description

PD-1 -REGULATED IL-2 IMMUNOCYTOKINE AND USES THEREOF

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 2, 2024, is named “51177-047WO2_Sequence_Listing_5_2_24” and is 197,852 bytes in size.

BACKGROUND

Interleukin 2 (IL-2) is a potent cytokine that exhibits toxicity upon systemic administration. There is a need for a version of IL-2 that can be delivered systemically but can be regulated to exhibit therapeutic activity on an effective subset of T cells.

SUMMARY

The present invention provides, inter alia, PD-1 -regulated IL-2 immunoconjugates (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or antibodies (e.g., including two DBA moieties or including two anti-PD-1 antibody moieties), compositions comprising the immunoconjugates and/or antibodies (e.g., pharmaceutical compositions), polynucleotides encoding the immunoconjugates and/or antibodies, vectors, host cells, methods of production, and methods and uses thereof.

In one aspect, the invention provides immunoconjugate comprising: (a) a first binding domain comprising: (i) an IL-2 polypeptide; (ii) a linker; and (iii) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus, and wherein the first binding domain is configured such that: (i) when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor; and (ii) when the DBA is bound to PD-1 , the DBA moiety is substantially blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to an IL-2 receptor; and (b) a second binding domain comprising an anti-PD-1 antibody moiety comprising a VH and a VL.

In some aspects, the anti-PD-1 antibody moiety does not substantially bind to an IL-2 polypeptide.

In some aspects, the DBA moiety comprises a Fab molecule. In some aspects, the DBA moiety comprises a Fab heavy chain comprising the VH of the DBA moiety and a heavy chain constant domain 1 (CH1 ) and a Fab light chain comprising the VL of the DBA moiety and a light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other. In some aspects, the DBA moiety is a conventional Fab molecule.

In some aspects, the anti-PD-1 antibody moiety comprises a Fab molecule. In some aspects, the anti-PD-1 antibody moiety comprises a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

In some aspects, (a) the DBA moiety is a conventional Fab molecule; and (b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

In some aspects, (a) the DBA moiety is a conventional Fab molecule; and (b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other.

In some aspects, the anti-PD-1 antibody moiety is a conventional Fab molecule.

In some aspects, (a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and (b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

In some aspects, (a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and (b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

In some aspects, the anti-PD-1 antibody moiety is a single-chain variable fragment (scFv).

In some aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VH-VL-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus. In other aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VL-VH-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus. In some aspects, the VH and VL of the scFv are connected by a linker. In some aspects, the VH, VL, and linker are connected in the following orientation: N-VH-linker-VL-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus. In other aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VL-linker-VH-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus.

In some aspects, the immunoconjugate further comprises an Fc domain comprising a first subunit and a second subunit. In some aspects, (a) the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus; and/or (b) the anti-PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

In some aspects, the Fc domain is an IgG Fc domain. In some aspects, the IgG Fc domain is an IgG 1 Fc domain. In some aspects, the Fc domain is a human IgG Fc domain.

In some aspects, the first subunit comprises one or more CH domains selected from a first CH2 (CH2i) domain and/or a first CH3 (CH3i) domain; and the second subunit comprises one or more CH domains selected from a second CH2 (CH22) domain and/or a second CH3 (CH32) domain. In some aspects, at least one of the one or more CH domains is paired with another CH domain. In some aspects, the CH3i and CH32 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH3i domain is positionable in the cavity or protuberance, respectively, in the CH32 domain. In some aspects, the CH3i and CH32 domains meet at an interface between the protuberance and cavity. In some aspects, the CH2i and CH22 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH2i domain is positionable in the cavity or protuberance, respectively, in the CH22 domain. In some aspects, the CH2i and CH22 domains meet at an interface between the protuberance and cavity.

In some aspects, (a) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (b) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index). In some aspects, the first subunit and/or the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index).

In some aspects, the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1 . In some aspects, the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1 .

In some aspects, binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and/or binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 or binding of the anti-PD- 1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 . In some aspects, (a) binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 ; or (b) binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD- 1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD- L1.

In some aspects, the DBA moiety comprises the following six complementarity-determining regions (CDRs): (a) a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR- H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); or (b) a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75).

In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; (b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; or (c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 ; or (d) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 .

In some aspects, the DBA moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; (b) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 76; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or (d) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 91 .

In some aspects, the anti-PD-1 antibody moiety comprises the following six CDRs: (a) a CDR- H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); (b) a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR- H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39); or (c) a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR-H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3).

In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40; or (c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising the amino acid sequence of SEQ ID NO: 22; (b) a VH comprising the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising the amino acid sequence of SEQ ID NO: 40; or (c) a VH comprising the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising the amino acid sequence of SEQ ID NO: 4.

In some aspects, (a) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); (b) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); or (c) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39).

In some aspects, (a) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (b) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; (c) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; or (d) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40.

In some aspects, (a) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti- PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22; (b) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22; (c) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or (d) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 40.

In some aspects, the linker is between 5 to 30 amino acids in length. In some aspects, the linker is 20 amino acids in length. In some aspects, the linker comprises the amino acid sequence (G2SG2)X, wherein x is an integer between 1 and 6 (SEQ ID NOs: 149-154). In some aspects, x is 2 (SEQ ID NO: 150) or 4 (SEQ ID NO: 152).

In some aspects, the IL-2 polypeptide is connected to the VH of the DBA moiety through the linker. In some aspects, the IL-2 polypeptide is connected to the VH of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C, wherein N- denotes a polypeptide N- terminus, and -C denotes a polypeptide C-terminus.

In other aspects, the IL-2 polypeptide is connected to the VL of the DBA moiety through the linker. In some aspects, the IL-2 polypeptide is connected to the VL of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C, wherein N- denotes a polypeptide N- terminus, and -C denotes a polypeptide C-terminus.

In some aspects, the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, and/or an alanine residue at position 125 (numbered relative to human IL-2 sequence of SEQ ID NO: 147). In some aspects, the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, an alanine residue at position 125, and/or a threonine residue at position 126 (numbered relative to human IL-2 sequence of SEQ ID NO: 147). In some aspects, the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 146. In some aspects, the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 199. In some aspects, the PD-1 is human PD-1 .

In one aspect, the invention provides an immunoconjugate comprising: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and (Hi) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein: (a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or (b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule. In one aspect, the invention provides an immunoconjugate comprising: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (iii) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other.

In one aspect, the invention provides an immunoconjugate comprising: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and (iii) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]- C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD- 1 antibody moiety is an scFv comprising a VH and a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the anti-PD- 1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]- [second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C- terminus.

In some aspects, the immunoconjugate comprises: (a) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (b) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (c) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; (d) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; (e) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; (f) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120; (g) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128; (h) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; or (i) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.

In some aspects, the immunoconjugate comprises: (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (c) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; (d) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128; (e) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120; (f) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120; (g) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128; (h) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; or (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

In one aspect, the invention provides an immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

In one aspect, the invention provides an isolated polynucleotide or a set of isolated polynucleotides encoding any of the immunoconjugates described herein.

In one aspect, the invention provides a vector or a set of vectors comprising any one of the isolated polynucleotides or and one of the sets of isolated polynucleotides described herein.

In one aspect, the invention provides a host cell or a set of host cells comprising (i) any of the isolated polynucleotide or any of the set of isolated polynucleotides described herein or (ii) the vector or the set of vectors described herein.

In one aspect, the invention provides a method of producing an immunoconjugate, comprising the steps of (a) culturing any one of the host cells or any one of the sets of host cells described herein under conditions suitable for the expression of the immunoconjugate. In some aspects, the method further comprises recovering the immunoconjugate.

In some aspects, the host cell expresses the first binding domain and the second binding domain. In some aspects, a first host cell expresses the first binding domain, and a second host cell expresses the second binding domain.

In some aspects, the method further comprises recovering the first binding domain and the second binding domain. In some aspects, the method further comprises contacting the recovered first binding domain with the recovered second binding domain.

In one aspect, the invention provides an immunoconjugate produced by any one of the methods described herein.

In one aspect, the invention provides a pharmaceutical composition comprising any one of the immunoconjugates described herein and a pharmaceutically acceptable carrier.

In one aspect, the invention provides any one of the immunoconjugates or pharmaceutical compositions described herein for use as a medicament.

In one aspect, the invention provides use of any one of the immunoconjugates or pharmaceutical compositions described herein in the manufacture of a medicament.

In one aspect, the invention provides any one of the immunoconjugates or pharmaceutical compositions described herein for use in the treatment of a cancer in a subject in need thereof.

In one aspect, the invention provides use of any one of the immunoconjugates or pharmaceutical compositions described herein in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof.

In one aspect, the invention provides use of any one of the immunoconjugates or pharmaceutical compositions described herein for treating a cancer in a subject in need thereof. In one aspect, the invention provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of any one of the immunoconjugates or pharmaceutical compositions described herein.

In some aspects, the cancer is a PD-1 -positive cancer.

In some aspects, the immunoconjugate for use, pharmaceutical composition for use, use, or method described herein further comprises administering an additional therapeutic agent to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a PD-1/PD-L1 blockade bioassay for different PD-1 binders. 7G12 constructs include the anti-PD-1 moiety AB003058. The “Always on” construct contains 0376 binders. Isotype is a negative control.

FIG. 2 shows potency and cis-/trans-signaling of a PD-1 -regulated IL-2 immunoconjugate as IL-2R signaling. PD-1 -IL-2v: always-on PD-1 -IL-2 construct (/.e., “always-on P1 AE4422-14542”); FAP-IL-2V: untargeted IL-2 polypeptide; AF5842: PD-1 -regulated IL-2 immunoconjugate. Preblocked: preblocked with competing anti-PD-1 .

FIG. 3A and FIG. 3B show potency and cis-/trans-signaling of various immunoconjugates in activating IL-2 receptor (IL-2R) signaling. FIG. 3A is a graph showing the results of the assay. FIG. 3B shows structures and orientation of the molecules tested. Crescent/circle in Fc region shows knob-in-hole modification. Solid circle shows IL-2 polypeptide. Un-patterned VH/VL indicate DBA moieties. Patterned VH/VL indicate anti-PD-1 antibody moieties. bl.: blocking.

FIG. 4A and FIG. 4B show potency and cis-/trans-signaling of various immunoconjugates in activating IL-2R signaling. FIG. 4A is a graph showing the results of the assay. FIG. 4B shows structures and orientation of the molecules tested. Crescent/circle in Fc region shows knob-in-hole modification. Solid circle shows IL-2 polypeptide. Un-patterned VH/VL indicate DBA moieties. Stripe-patterned VH/VL indicate anti-PD-1 antibody moieties. Checker-patterned VH/VL indicates non-blocking DBA moieties. For each pair of cylinders representing VH/VL or CH1/CL: the darker- colored cylinder represents VH or CH1 and the lighter-colored cylinder represents CL or CH1 . b or bl.: blocking; nb: non-blocking.

FIG. 5A and FIG. 5B show potency and cis-/trans-signaling of various immunoconjugates in activating IL-2R signaling. FIG. 5A is a graph showing the results of the assay. FIG. 5B shows structures and orientation of the molecules tested. Crescent/circle in Fc region shows knob-in-hole modification. Solid circle shows IL-2 polypeptide. Un-patterned VH/VL indicate DBA moieties. Stripe-patterned VH/VL indicate anti-PD-1 antibody moieties. Checker-patterned VH/VL indicates non-blocking DBA moieties. For each pair of cylinders representing VH/VL or CH1/CL: the darker- colored cylinder represents VH or CH1 and the lighter-colored cylinder represents CL or CH1 . P1 AI7462 comprises an scFV for the anti-PD-1 antibody moiety, b or bl.: blocking; nb: non-blocking.

FIG. 6 shows potency and cis-/trans-signaling of PD-1 -regulated IL-2 immunoconjugates in activating IL-2R signaling, bl.: blocking FIG. 7A and FIG. 7B show the ability of various immunoconjugates at eliciting cytotoxic T cell effector function. Minimal MLR: minimal mixed lymphocyte reaction. FIG. 7A illustrates the assay. FIG. 7B shows results of the assay for different immunoconjugate constructs.

FIG. 8A and FIG. 8B show the ability of PD-1 -regulated IL-2 immunoconjugates to rescue conventional T_COnv effector functions from T_reg suppression. FIG. 8A illustrates the assay. FIG. 8B shows the results of the rescue assay. Each symbol represents a separate donor, horizontal lines indicate medians with N = 9 donors, from 3 independent experiments. P was calculated using oneway ANOVA (*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 ).

FIG. 9A - FIG. 9C show potency and cis-/trans-signaling of various immunoconjugates in activating IL-2R signaling. FIG. 9A illustrates the assay. FIG. 9B shows structures and orientation of the molecules tested. Crescent/circle in Fc region shows knob-in-hole modification. Solid circle shows IL-2 polypeptide. Un-patterned VH/VL indicates DBA moieties. Stripe-patterned VH/VL indicates anti-PD-1 antibody moieties. Checker-patterned VH/VL indicates non-blocking DBA moieties. For each pair of cylinders representing VH/VL or CH1/CL: the darker-colored cylinder represents VH or CH1 and the lighter-colored cylinder represents CL or CH1 . FIG. 9C shows the results of the assay. In particular, as compared to P1 AI7440, P1 AJ1837 only differs by having an extra Asp residue at the C-terminus of the DBA light chain. The same applies to P1 AJ1838 (one extra C-terminal Asp residue) and P1 AI7441 . b: blocking; nb: non-blocking; dei: de-immunized.

FIG. 10A - FIG. 10D show potency, cis-/trans-signaling, and leakiness of various immunoconjugates for IL-2R signaling, e.g., on activated CD4 cells (FIG. 10A), activated CD8 cells (FIG. 10B), and NK cells (FIG. 10C). FIG. 10D shows structures and orientation of the molecules tested. Crescent/circle in Fc region shows knob-in-hole modification. Solid circle shows IL-2 polypeptide. Un-patterned VH/VL indicates DBA moieties. Stripe-patterned VH/VL indicates anti-PD- 1 antibody moieties. Checker-patterned VH/VL indicates non-blocking DBA moieties. For each pair of cylinders representing VH/VL or CH1/CL: the darker-colored cylinder represents VH or CH1 and the lighter-colored cylinder represents CL or CH1 . B: blocking; nb: non-blocking; dei: de-immunized.

FIG. 11 A and FIG. 11B show results of binding competition assay to determine whether blocking DBA moieties (FIG. 11A) or non-blocking DBA moieties (FIG. 11A) of a PD-1 -regulated IL-2 immunoconjugate competes for PD-1 binding with a blocking or non-blocking anti-PD-1 antibody moiety arm. B: blocking; nb: non-blocking; mat: affinity matured.

FIG. 12A and FIG. 12B show results of enzyme-linked immunosorbent assay (ELISA)-based assay to assess switching behavior of PD-1 -regulated IL-2 immunoconjugates. FIG. 12A shows PD-1 concentration-dependent switching of immunoconjugates. FIG. 12B further provides IgG concentration-dependent negative control for each tested immunoconjugate.

FIG. 13A - FIG. 13M show results of HEK-Blue IL-2 reporter assay, showing PD-1 concentration-dependent and PD-1 -regulated IL-2 immunoconjugate concentration-dependent signal. Each panel shows results for a different immunoconjugate. FIG. 13A: always-on P1 AE4422-14542 control; FIG. 13B: P1 AI7455; FIG. 13C: P1 AI7441 ; FIG. 13D: P1AI7465; FIG. 13E: P1AI7476; FIG. 13F: P1 AI7440; FIG. 13G: P1AI7464; FIG. 13H: P1 AI7474; FIG. 131: P1AI7438; FIG. 13J: P1 AI7462; FIG. 13K: P1AI7473; FIG. 13L: P1AI7443; FIG. 13M: P1 AI7467.

FIG. 14 shows in vivo efficacy of PD-1 -regulated IL-2 immunoconjugates as single agents at inhibiting pancreatic tumor growth in a syngeneic model using mouse pancreatic Panc02-Fluc cell line.

FIG. 15 shows lung weight of mice treated with vehicle, PD-1 -regulated IL-2 immunoconjugate, or pembrolizumab as a metric for adverse effects of treatment.

FIG. 16A and FIG. 16B show the impact of PD-1 -regulated IL-2 immunoconjugates on CD8⁺ T cell expansion in tumor (FIG. 16A) and blood (FIG. 16B).

FIG. 17A and FIG. 17B show potency and cis-/trans-signaling of PD-1 -regulated IL-2 immunoconjugates and control in activating IL-2R signaling. FIG. 17A compares the 1 +1 format of the PD-1 -reg-IL-2v with one DBA and one PD-1 only binder, in which IL-2v is attached to the DBA to the asymmetric format of Compound C with two DBAs, in which IL-2v is attached to one DBA. FIG. 17B compares PD-1 -reg-IL-2v with PD-1 -reg-IL-2vQ126T, which contains an additional mutation in the IL-2v to further decrease the binding affinity for IL-2Rb and therefore increase the conditional activity of the IL-2v on the PD-1 expression. Data shown as mean ± SEM of 4 donors.

FIG. 18A - FIG. 18C show potency, cis-/trans-signaling, and leakiness of various immunoconjugates for IL-2R signaling, e.g., on activated CD4 cells (FIG. 18A), activated CD8 cells (FIG. 18B), and NK cells (FIG. 18C). Data show mean ± SEM of 2 donors.

FIG. 19 shows IL-2 stimulation of JAK/STAT5 activation by different PD-1 -regulated IL-2 immunoconjugates and the subsequent secretion of SEAP as detected by HEK-blue™ IL-2 reporter cell line. Activation of reporter cell lines was detected as absorbance change at 640 nm.

FIG. 20 shows potency and cis-/trans-signaling of PD-1 -regulated IL-2 immunoconjugates with 10-mer or 20-mer linkers between the DBA and the IL-2 polypeptide. Data shown as mean ± SEM of 4 donors.

DETAILED DESCRIPTION

The present invention is based, at least in part, on Applicant’s discovery that the PD-1 - regulated IL-2 immunoconjugates as disclosed herein are unexpectedly advantageous for targeting IL-2-mediated cytotoxicity to PD-1 -rich cellular environments (e.g., cells expressing PD-1 ). IL-2 is a potent cytokine that exhibits toxicity upon systemic administration. However, the PD-1 -regulated IL-2 immunoconjugates described herein allow for targeted administration of IL-2 for treatment, e.g., of cancer. In particular, the present immunoconjugates show surprising sensitivity and discrimination for directing IL-2 signaling in the presence versus absence of PD-1 . Thus, the present invention provides for improved therapeutic options for targeted treatment of cancer.

The invention is also based, at least in part, on Applicant’s discovery that the structure and orientation of PD-1 -regulated IL-2 immunoconjugates as disclosed herein exhibit superior therapeutic effects compared to other tested structures and orientations of immunoconjugates. For example, immunoconjugates as disclosed herein may include a first binding domain that includes an IL-2 polypeptide and a dual-binding antibody (DBA) moiety that binds to PD-1 and the IL- 2 polypeptide, e.g., in a mutually exclusive manner, and a second binding domain that includes an anti-PD-1 antibody. In some examples, use of a monospecific anti-PD-1 antibody moiety in the second binding domain in immunoconjugates as disclosed herein {e.g., as a targeting arm) can result in superior efficacy {e.g., in terms of anti-tumor activity) and safety compared to constructs that include only a DBA that binds to PD-1 and an IL-2 polypeptide {e.g., a bivalent construct that includes two DBA moieties). For example, such constructs as disclosed herein can have reduced leakiness in terms of IL-2 unmasking, resulting in less toxicity.

The invention is also based, at least in part, on Applicant’s discovery that the structure and orientation of PD-1 -regulated IL-2 immunoconjugates as disclosed herein can allow for improved developability and ease of manufacturing compared to other tested structures and orientations of immunoconjugates. For example, the PD-1 -regulated IL-2 immunoconjugates as disclosed herein can be less prone to aggregation compared to other structures and orientations of immunoconjugates.

I. DEFINITIONS

An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

By “administering” is meant a method of giving a dosage of a compound {e.g., a PD-1 - regulated IL-2 immunoconjugate or antibody disclosed herein) or a composition {e.g., a pharmaceutical composition, e.g., a pharmaceutical composition including a PD-1 -regulated IL-2 immunoconjugate or antibody disclosed herein) to a subject. The compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The method of administration can vary depending on various factors {e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule {e.g., an antibody) and its binding partner {e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair {e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are also described herein.

An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementary determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

The terms “anti-PD-1 antibody” and “an antibody that binds to PD-1” refer to an antibody that is capable of binding PD-1 with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting PD-1 . In one aspect, the extent of binding of an anti-PD-1 antibody to an unrelated, non-PD-1 protein is less than about 10% of the binding of the antibody to PD-1 as measured, e.g., by surface plasmon resonance (SPR). In certain aspects, an antibody that binds to PD-1 has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM {e.g., 10^-8 M or less, e.g., from 10^-13 M to 10^-8 M, e.g., from 10^-13 M to 10^-9 M). In certain aspects, an antibody that binds to PD-1 has a KD of from about 1 x 10¹² M to about 1 x 10 M, from about 1 x 10^-12 M to about 1 x 10^-11 M, or from about 1 x 10^-11 M to about 5 x 10^-11 M. An antibody is said to “specifically bind” to PD-1 when the antibody has a KD of 1 pM or less.

The terms “dual binding antibody” or “DBA” refer to an antibody that is capable of binding two different antigens {e.g., PD-1 and IL-2) with sufficient affinity in a mutually exclusive manner. In one embodiment, the extent of binding of a DBA to an unrelated, non-targeted protein {e.g., other than the two target antigens) is less than about 10% of the binding of the antibody to the two target antigens {e.g., PD-1 and IL-2) as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, a DBA has a dissociation constant (Kd) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM {e.g. 10⁸ M or less, e.g., from 10⁸ M to 10 ¹³ M, e.g., from 10⁹ M to 10 ¹³ M).

The term “antibody” herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies {e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules {e.g., scFv, and scFab); single domain antibodies (dAbs); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology (2005) 23:1126-1136. By “binding domain” or “binding moiety” is meant a part of a compound or a molecule that specifically binds to a target epitope, antigen, ligand, or receptor. Molecules featuring binding moieties include, but are not limited to, antibodies {e.g., monoclonal, polyclonal, recombinant, humanized, and chimeric antibodies), antibody fragments or portions thereof {e.g., Fab fragments, Fab’2, scFv antibodies, SMIP, domain antibodies, diabodies, minibodies, scFv-Fc, affibodies, nanobodies, and VH and/or VL domains of antibodies), receptors, ligands, aptamers, and other molecules having an identified binding partner. In particular aspects, molecules featuring binding domains include immunoconjugates, e.g., PD-1 -regulated IL-2 immunoconjugates.

As used herein, the terms “first,” “second,” or “third,” with respect to Fc subunits and the like, are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly so stated.

A “Fab molecule” refers to a protein that includes or consists of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

By “fused” or “linked” is meant that two moieties {e.g., a DBA moiety and an IL-2 polypeptide or a VH and a VL of an scFv) are linked by a covalent bond, e.g., by a peptide bond, either directly or via one or more peptide linkers. A “linker” as used herein refers to a short polypeptide (e.g., comprising 5-40 amino acids; e.g., comprising 5-30 amino acids; e.g., comprising 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 amino acids) used to link two other moieties {e.g., aDBA moiety and an IL-2 polypeptide or a VH and a VL of an scFv).

As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the binding domains {e.g., the anti- PD-1 antibody moiety) of an immunoconjugate includes a single-chain Fab molecule, i.e., a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

A “single-chain variable fragment” or “scFv” is a fusion protein of the variable domains of the heavy (VH) and light chains (VL) of an antibody, connected by a linker. In particular, the linker is a short polypeptide, typically of 10 to 25 amino acids and is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C- terminus of the VL, or vice versa. This protein retains the specificity of the original antibody, despite removal of the constant regions and the introduction of the linker. For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571 ,894 and 5,587,458.

A “single chain Fab fragment” or “scFab” is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1 ), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1 -linker-VL-CL, b) VL-CL-linker-VH-CH1 , c) VH-CL-linker-VL-CH1 or d) VL-CH1 - linker-VH-CL. In particular, said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. Said single chain Fab fragments are stabilized via the natural disulfide bond between the CL domain and the CH1 domain. In addition, these single chain Fab fragments might be further stabilized by generation of interchain disulfide bonds via insertion of cysteine residues {e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

By a “crossover” Fab molecule (also termed “Crosstab”) is meant a Fab molecule wherein the variable domains of the Fab heavy and light chain are exchanged {i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 , in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH- CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the “heavy chain” of the crossover Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fab molecule in its natural format, i.e., comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CH1 , in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

“Diabodies” are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161 ; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger etal., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

“Single-domain antibodies” are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain aspects, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1 ).

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Aspects of cancer include solid tumor cancers and non-solid tumor cancers. Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, bladder cancer {e.g., urothelial carcinoma (UC), including metastatic UC (mUC); muscle-invasive bladder cancer (MIBC), and non-muscle- invasive bladder cancer (NMIBC)); kidney or renal cancer {e.g., renal cell carcinoma (RCC)); lung cancer, including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; cancer of the urinary tract; breast cancer {e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), which are estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-) negative); prostate cancer, such as castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer {e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer {e.g., hepatocellular carcinoma (HCC)); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade/follicu lar non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs’ syndrome, brain cancer, head and neck cancer, and associated metastases.

“Tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder,” and “tumor” are not mutually exclusive as referred to herein.

The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In another embodiment, the cell proliferative disorder is a tumor.

The term “epitope” denotes the site on an antigen, either proteinaceous or non-proteinaceous, to which a DBA moiety, an anti-PD-1 antibody moiety, a PD-1 -regulated IL-2 immunoconjugate, or an antibody described herein binds. Epitopes can be formed both from contiguous amino acid stretches (linear epitope) or comprise non-contiguous amino acids (conformational epitope), e.g., coming in spatial proximity due to the folding of the antigen, i.e., by the tertiary folding of a proteinaceous antigen. Linear epitopes are typically still bound by a DBA moiety, an anti-PD-1 antibody moiety, a PD-1 -regulated IL-2 immunoconjugate, or an antibody described herein after exposure of the proteinaceous antigen to denaturing agents, whereas conformational epitopes are typically destroyed upon treatment with denaturing agents. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 10, at least 15, at least 20, at least 30, or at least 35, or 3-25, 3-20, 3- 15, 3-10, 3-5, 30-40, 35-40, or 5-10 amino acids in a unique spatial conformation.

Screening for antibodies binding to a particular epitope {i.e., those binding to the same epitope) can be done using methods routine in the art such as, e.g., without limitation, alanine scanning, peptide blots (see, e.g., Kobeissy et al., Meth. Mol. Biol. (2004) 248: 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see, e.g., Hochleitner et al., Prot. Sci. 9 (2000) 487-496), and cross-blocking (see, e.g., “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

Competitive binding can be used to easily determine whether an antibody or DBA binds to the same epitope of PD-1 as, or competes for binding with, a reference anti-PD-1 antibody or DBA that binds PD-1 . For example, an “antibody that binds to the same epitope” as a reference anti-PD-1 antibody or DBA refers to an antibody or DBA that blocks binding of the reference anti-PD-1 antibody or DBA to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more. Also for example, to determine if an antibody binds to the same epitope as a reference anti-PD-1 antibody or DBA, the reference antibody or DBA is allowed to bind to PD-1 under saturating conditions. After removal of the excess of the reference antibody, the ability of an anti-PD-1 antibody or DBA in question to bind to PD-1 is assessed. If the anti-PD-1 antibody or DBA is able to bind to PD-1 after saturation binding of the reference anti-PD-1 antibody or DBA, it can be concluded that the anti-PD-1 antibody or DBA in question binds to a different epitope than the reference anti-PD-1 antibody or DBA. But, if the anti-PD-1 antibody or DBA in question is not able to bind to the target epitope after saturation binding of the reference anti-PD-1 antibody or DBA, then the anti-PD-1 antibody or DBA in question may bind to the same epitope as the epitope bound by the reference anti-PD-1 antibody or DBA.

In some aspects, two antibodies or DBAs are deemed to bind to the same or an overlapping epitope if a 1 -, 5-, 10-, 20-, or 100-fold excess of one antibody or DBA inhibits binding of the other by at least 50%, at least 75%, at least 90% or even 99% or more as measured in a competitive binding assay (see, e.g., Junghans etal., Cancer Res. 50 (1990) 1495-1502).

In some aspects, two antibodies or DBAs are deemed to bind to the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody or DBA also reduce or eliminate binding of the other. Two antibodies or DBAs are deemed to have “overlapping epitopes” if only a subset of the amino acid mutations that reduce or eliminate binding of one antibody or DBA reduce or eliminate binding of the other.

The term “chimeric” antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, lgG2, IgGs, lgG4, IgAi, and lgA2. In certain aspects, the antibody is of the IgG 1 isotype. In certain aspects, the antibody is of the IgG 1 isotype with the P329G, L234A and L235A mutations to reduce Fc-region effector function. In other aspects, the antibody is of the lgG2 isotype. In certain aspects, the antibody is of the lgG4 isotype with the S228P mutation in the hinge region to improve stability of lgG4 antibody. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, Y, and p, respectively. The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain.

The terms “constant region derived from human origin” or “human constant region” as used herein denotes a constant heavy chain region of a human antibody of the subclass IgG 1 , lgG2, lgG3, or lgG4 and/or a constant light chain kappa or lambda region. Such constant regions are well known in the state of the art and e.g., described by Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991 ) (see also e.g., Johnson, G., and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218; Kabat, E.A., et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, numbering of amino acid residues in the constant region is according to the EU numbering system, also called the EU index of Kabat, as described in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991 ), NIH Publication 91 -3242.

“Effector functions” refer to those biological activities attributable to the Fc region of a PD-1 - regulated IL-2 immunoconjugate or antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors {e.g., B cell receptor); and B cell activation.

An “effective amount” of a compound, for example, a PD-1 -regulated IL-2 immunoconjugate or antibody disclosed herein or a composition {e.g., pharmaceutical composition) thereof, is at least the minimum amount required to achieve the desired therapeutic or prophylactic result, such as a measurable improvement or prevention of a particular disorder {e.g., a cell proliferative disorder, e.g., cancer). An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting {i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit {i.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full- length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. In one aspect, a heavy chain including an Fc region as specified herein, comprised in a PD-1 -regulated IL-2 immunoconjugate or an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In one aspect, a heavy chain including an Fc region as specified herein, comprised in a PD-1 -regulated IL-2 immunoconjugate or an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to EU index).

“Framework” or “FR” refers to variable domain residues other than complementary determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the CDR and FR sequences generally appear in the following sequence in VH (or VL): FR1 -CDR-H1 (CDR-L1 )-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)- FR4.

The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein. It should be understood that the full-length antibody comprises a heavy chain variable domain and light chain variable domain, as defined herein, and an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells”, which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91 -3242, Bethesda MD (1991 ), vols. 1 -3. In one aspect, for the VL, the subgroup is subgroup kappa I as in Kabat et al., supra. In one aspect, for the VH, the subgroup is subgroup III as in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

The term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”).

In certain aspects, antibodies comprise six CDRs: three in the VH (CDR-H1 , CDR-H2, CDR- H3), and three in the VL (CDR-L1 , CDR-L2, CDR-L3). In certain aspects, the antibodies comprising six CDRs are full-length antibodies. In certain aspects, the antibodies comprising six CDRs are antibody fragments.

Exemplary CDRs herein include:

(a) hypervariable loops occurring at amino acid residues 26-32 (L1 ), 50-52 (L2), 91 -96 (L3), 26-32 (H1 ), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 -917 (1987));

(b) CDRs occurring at amino acid residues 24-34 (L1 ), 50-56 (L2), 89-97 (L3), 31 -35b (H1 ), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991 )); and

(c) antigen contacts occurring at amino acid residues 27c-36 (L1 ), 46-55 (L2), 89-96 (L3), 30- 35b (H1 ), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)). Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to McCallum, supra, Chothia, supra, or any other scientifically accepted nomenclature system.

A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals {e.g., cows, sheep, cats, dogs, and horses), primates {e.g., humans and non-human primates such as monkeys), rabbits, and rodents {e.g., mice and rats). In certain aspects, the subject is a human.

An “immunoconjugate” is an antibody {e.g., a DBA moiety and/or an anti-PD-1 antibody moiety) conjugated to one or more heterologous molecule(s), including but not limited to a polypeptide (e.g., an IL-2 polypeptide). In a particular aspect, the heterologous molecule is an IL-2 polypeptide, e.g., a human IL-2 polypeptide, e.g., a mutant human IL-2 polypeptide described herein.

A “PD-1 -regulated IL-2 immunoconjugate” or “PD-1 -reg-IL-2v” is an immunoconjugate comprising an IL-2 polypeptide, wherein the IL-2 polypeptide activity {e.g., activating signaling of IL- 2R), is regulated by an antigen-binding domain {e.g., which binds to PD-1 ) of the immunoconjugate. In some aspects, a PD-1 -regulated IL-2 immunoconjugate includes a DBA moiety that binds to PD-1 and IL-2 polypeptide in in a substantially mutually exclusive manner or in a mutually exclusive manner. A PD-1 -regulated IL-2 immunoconjugate may further include an anti-PD-1 antibody moiety, e.g., that does not substantially bind to IL-2 or that does not bind to IL-2.

An “isolated” immunoconjugate (e.g., PD-1 -regulated IL-2 immunoconjugate) or antibody is one which has been separated from a component of its natural environment. In some aspects, an immunoconjugate (e.g., an PD-1 -regulated IL-2 immunoconjugate) or an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic {e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic {e.g., ion exchange or reverse phase HPLC) methods. For a review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

The term “nucleic acid molecule” or “polynucleotide” includes any compound and/or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base {i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar {i.e., deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA; ribonucleic acid (RNA), in particular, messenger RNA (mRNA); synthetic forms of DNA or RNA; and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecules can contain naturally occurring or non- naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody as described herein in vitro and/or in vivo, e.g., in a host or subject. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al, Nature Medicine 2017, published online 12 June 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1 ).

An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

An “isolated nucleic acid encoding an immunoconjugate” or “isolated nucleic acid encoding an antibody” refers to one or more nucleic acid molecules encoding an immunoconjugate (e.g., a PD-1 - regulated IL-2 immunoconjugate) or antibody described herein, including heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies in accordance with the present disclosure may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phagedisplay methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

“Multispecific antigen-binding molecules” and “multispecific antibodies” are monoclonal antigen-binding molecules or antibodies, respectively, that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In particular embodiments, multispecific antibodies may be “bispecific antibodies,” which have binding specificity for two different sites, i.e., two different epitopes on two different antigens or two different epitopes on the same antigen.

“Native antibodies” refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide- bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant heavy domains (CH1 , CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain.

The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001/007611 .

Unless otherwise indicated, for purposes herein, percent amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; W. R. Pearson (1996) “Effective protein sequence comparison” Meth. Enzymol. 266:227- 258; and Pearson et. al. (1997) Genomics 46:24-36 and is publicly available from fasta.bioch.virginia.edu/fasta_www2/fasta_down.shtml or ebi.ac.uk/Tools/sss/fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu/fasta_ www2/index.cgi can be used to compare the sequences, using the ggsearch (global protei protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

The terms “pharmaceutical composition” and “pharmaceutical formulation” are used interchangeably herein, and refer to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the pharmaceutical composition would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “PD-1 ,” as used herein, refers to any native PD-1 from any vertebrate source, including mammals such as primates {e.g., humans, monkeys (cyno)), and rodents {e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed PD-1 as well as any form of PD-1 that results from processing in the cell. The term also encompasses naturally occurring variants of PD-1 , e.g., splice variants or allelic variants. In certain aspects, the PD-1 is a human PD-1. An exemplary human PD-1 is shown in Uni ProtKB/Swiss-Prot Accession No. Q15116.

The term “interleukin-2” or “IL-2,” as used herein, refers to any native IL-2 from any vertebrate source, including mammals such as primates {e.g., humans) and rodents {e.g., mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed IL-2, as well as any form of IL-2 that results from processing in the cell. The term also encompasses naturally occurring variants of IL-2, including, for example, splice variants or allelic variants. IL-2 includes, for example, human IL-2 {e.g., wildtype human IL-2), the polypeptide sequence of which is SEQ ID NO: 147. A “mutant” IL-2 used herein refers to an IL-2 comprising one or more amino acid modifications as compared to a wildtype IL-2 polypeptide sequence. In a particular aspect, a mutant human IL-2 polypeptide comprises one or more amino acid modifications as compared to the wildtype human IL-2 polypeptide of SEQ ID NO: 147. In a preferred embodiment, a mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 146. In a further preferred embodiment, a mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 199.

As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease {e.g., cancer) in the subject being treated, and can be performed either for prophylaxis (“preventative treatment” or “prophylactically treating”) or during the course of clinical pathology (“therapeutic treatment” or “therapeutically treating”). Desirable effects of therapeutic treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis of the cancer, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Desirable effects of preventative treatment include, but are not limited to, preventing occurrence or recurrence of disease. In some aspects, antibodies as described herein are used to delay development of a disease or to slow the progression of a disease.

The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementary determining regions (CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6^th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson etal., Nature 352:624-628 (1991 ).

The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

II. COMPOSITIONS AND METHODS

In one aspect, the present disclosure provides immunoconjugates {e.g., immunoconjugates including at least one binding domain that binds to PD-1 conjugated, either directly or via a linker, to an IL-2 polypeptide {e.g., a binding domain including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner); and at least one binding domain that includes an anti-PD-1 antibody moiety that binds to PD-1 ). In some embodiments, the immunoconjugates include a first binding domain including an IL-2 polypeptide, a linker, and a dual binding antibody (DBA) moiety and a second binding domain including an anti-PD-1 antibody moiety. The DBA moiety may include a heavy chain variable region (VH) and a light chain variable region (VL), and may bind to PD-1 and the IL-2 polypeptide in a mutually exclusive manner. In the first binding domain, the IL-2 polypeptide, the linker, and the DBA moiety may be connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus. The anti-PD-1 antibody moiety may include a VH and a VL. In some examples, the anti-PD-1 antibody moiety is monospecific. In some examples, the immunoconjugate is a PD-1 -regulated IL-2 immunoconjugate. Immunoconjugates and antibodies as described herein are useful, e.g., for the treatment of cancer.

A. Dual Binding Antibody (DBA) Moieties

In some aspects, immunoconjugates and antibodies described herein may include a binding domain {e.g., a first binding domain) that includes a DBA moiety. In certain aspects, a DBA moiety includes a heavy chain variable region (VH) and a light chain variable region (VL), and may bind to PD-1 and an IL-2 polypeptide, e.g., in a mutually exclusive manner. In certain aspects, the PD-1 is a human PD-1 . In certain aspects, the IL-2 polypeptide is a human IL-2 polypeptide or a modified human IL-2 polypeptide described herein. In some instances, the modified human IL-2 polypeptide contains one or more amino acids described herein {e.g., a human IL-2 comprising T3A, F42A, Y45A, L72G and C125A amino acid substitutions). In some aspects, a DBA moiety of the present invention includes at least one, at least two, at least three, at least four, at least five, or all six CDRs (e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1. In some instances, the DBA moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1 .

Table 1. Listing of SEQ ID NOs of DBA Moieties.

In some aspects, the invention provides a DBA moiety comprising the following six complementary-determining regions (CDRs): a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). In some aspects, the DBA moiety is AB002345.

In some aspects, the DBA moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 69; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 70; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 71 ; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 72; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 60; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 61 ; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 62; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 63. In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 67; (b) a VL comprising the amino acid sequence of SEQ ID NO: 58; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety is AB002345.

In some aspects, the invention provides a DBA moiety comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75). In some aspects, the DBA moiety is AB003637, AB003637 de-immunized, or AB003637 de-immunized2.

In some aspects, the DBA moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 87; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 89; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 90; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 80; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 81 . In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising the amino acid sequence of SEQ ID NO: 76; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety is AB003637.

In some aspects, the DBA moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 87; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 89; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 90; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 80; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 155. In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety is AB003637 de-immunized.

In some aspects, the DBA moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 87; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 156; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 90; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 80; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 155. In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 111 ; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 111 ; (b) a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, the DBA moiety is AB003637 de-immunized2.

In some aspects, the DBA moiety is a conventional Fab molecule. In some aspects, the DBA moiety is a conventional Fab molecule including a Fab heavy chain composed of the heavy chain variable and constant domains (VH-CH1 , in N- to C-terminal direction), and a Fab light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

In some aspects, the DBA moiety is a crossFab (/.e., is a Fab molecule including a crossover modification), wherein the DBA moiety includes a Fab heavy chain including the VH of the DBA moiety and a heavy chain constant domain 1 (CH1 ) and a Fab light chain including the VL of the DBA moiety and a light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other. In a preferred embodiment when the DBA moiety is a crossFab, the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

B. Anti-PD-1 Antibody Moieties

In some aspects, immunoconjugates and antibodies described herein may include a binding domain {e.g., a second binding domain) that includes an anti-PD-1 antibody moiety. In certain aspects, an anti-PD-1 antibody moiety includes a heavy chain variable region (VH) and a light chain variable region (VL), and binds to PD-1 . In certain aspects, the PD-1 is a human PD-1 . In some aspects, the anti-PD-1 antibody moiety does not substantially bind to an IL-2 polypeptide, e.g., an IL-2 polypeptide described herein. In some aspects, the PD-1 antibody moiety is monospecific.

In some aspects, an anti-PD-1 antibody moiety of the present invention includes at least one, at least two, at least three, at least four, at least five, or all six CDRs {e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2. In some instances, the anti-PD-1 antibody moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2. Table 2. Listing of SEQ ID NOs of Anti-PD-1 Moieties.

In some aspects, the invention provides an anti-PD-1 antibody moiety comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR- H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3). In some aspects, the anti-PD-1 antibody moiety is AB003058.

In some aspects, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 16; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 18; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 7; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 9. In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 13; (b) a VL comprising the amino acid sequence of SEQ ID NO: 4; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety is AB003058.

In some aspects, the invention provides an anti-PD-1 antibody moiety comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR- H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, the anti-PD-1 antibody moiety is 0376 deimmunized.

In some aspects, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 36; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 27. In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31 ; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 31 ; (b) a VL comprising the amino acid sequence of SEQ ID NO: 22; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety is 0376 de-immunized.

In some aspects, the invention provides an anti-PD-1 antibody moiety comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 166), a CDR-H2 comprising the amino acid sequence of TISGGGRDIYYPDSVKG (SEQ ID NO: 167), a CDR- H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 168), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 157), a CDR-L2 comprising the amino acid sequence of RSSTLES (SEQ ID NO: 158), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 159). In some aspects, the anti-PD-1 antibody moiety is 0376.

In some aspects, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 171 ; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 172; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 173; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 174; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 162; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 163; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 164; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 165. In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 169; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 160; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti- PD-1 antibody moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 169; (b) a VL comprising the amino acid sequence of SEQ ID NO: 160; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety is 0376.

In some aspects, the invention provides an anti-PD-1 antibody moiety comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR- H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39). In some aspects, the anti-PD-1 antibody moiety is 1040 affinity-matured.

In some aspects, the anti-PD-1 antibody moiety comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 51 ; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 54; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 42; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 43; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 44; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 45. In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 49; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 49; (b) a VL comprising the amino acid sequence of SEQ ID NO: 40; or (c) a VH as in (a) and a VL as in (b). In some aspects, the anti-PD-1 antibody moiety is 1040 affinity-matured.

In some aspects, the anti-PD-1 antibody moiety is a conventional Fab molecule. In some aspects, the anti-PD-1 antibody moiety is a conventional Fab molecule including a Fab heavy chain composed of the heavy chain variable and constant domains (VH-CH1 , in N- to C-terminal direction), and a Fab light chain composed of the light chain variable and constant domains (VL-CL, in N- to C- terminal direction).

In some aspects, the anti-PD-1 antibody moiety is a crossFab (/.e., is a Fab molecule including a crossover modification), wherein the anti-PD-1 antibody moiety includes a Fab heavy chain including the VH of the anti-PD-1 antibody moiety and a heavy chain constant domain 1 (CH1 ) and a Fab light chain including the VL of the anti-PD-1 antibody moiety and a light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other. In a preferred embodiment when the anti-PD-1 antibody moiety is a crossFab, the VH of the Fab heavy chain and VL of the Fab light chain are replaced by each other.

In some aspects, the anti-PD-1 antibody moiety is a single-chain variable fragment (scFv). In some aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VH-VL-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus. In other aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VL-VH-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus. In some aspects, the VH and VL of the scFv are connected by a linker. Any suitable linker, e.g., any linker described herein (e.g., in Section D below) may be used. In some aspects, the VH, VL, and linker are connected in the following orientation: N- VH-linker-VL-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C- terminus. In other aspects, the anti-PD-1 antibody is an scFv wherein the VH and VL are connected in the following orientation: N-VL-linker-VH-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

C. IL-2 polypeptides

The immunoconjugates of the present invention may include an IL-2 polypeptide {e.g., a human IL-2 polypeptide; e.g., a mutant IL-2 polypeptide; e.g., a mutant human IL-2 polypeptide) having advantageous properties for immunotherapy. In particular, pharmacological properties of an IL-2 polypeptide that contribute to toxicity but are not essential for efficacy of the IL-2 polypeptide may be eliminated in a mutant IL-2 polypeptide. Such mutant IL-2 polypeptides are described in detail in WO 2012/107417, which is incorporated herein by reference in its entirety. Different forms of the IL-2 receptor consist of different subunits and exhibit different affinities for IL-2. The intermediate-affinity IL-2 receptor, consisting of the p and y receptor subunits, is expressed on resting effector cells and is sufficient for IL-2 signaling. The high-affinity IL-2 receptor, additionally comprising the a-subunit of the receptor, is mainly expressed on regulatory T (T_reg) cells as well as on activated effector cells where its engagement by an IL-2 polypeptide can promote T_reg cell-mediated immunosuppression or activation-induced cell death (AICD), respectively. Thus, without wishing to be bound by theory, reducing or abolishing the affinity of an IL-2 polypeptide to the a-subunit of the IL-2 receptor can reduce IL-2-induced downregulation of effector cell function by regulatory T cells and development of tumor tolerance by the process of AICD. On the other hand, maintaining the affinity to the intermediate-affinity IL-2 receptor can preserve the induction of proliferation and activation of effector cells such as NK and T cells by the IL-2 polypeptide.

A mutant interleukin-2 (IL-2) polypeptide included in the immunoconjugate described herein may include at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor and preserves affinity of the mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor each compared to a wild-type IL-2 polypeptide.

Mutants of human IL-2 polypeptide (hlL-2 polypeptide) with decreased affinity to CD25 may for example be generated by amino acid substitution at amino acid position 35, 38, 42, 43, 45 or 72 or combinations thereof (numbering relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). Exemplary amino acid substitutions include K35E, K35A, R38A, R38E, R38N, R38F, R38S, R38L, R38G, R38Y, R38W, F42L, F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, K43E, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Particular mutant IL-2 polypeptides useful in the immunoconjugates described herein include an amino acid mutation at an amino acid position corresponding to residue 42, 45, or 72 of human IL-2 polypeptide, or a combination thereof. In one aspect, the amino acid mutation is an amino acid substitution selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, more specifically an amino acid substitution selected from the group of F42A, Y45A and L72G. These mutant IL-2 polypeptides exhibit substantially similar binding affinity to the intermediate-affinity IL-2 receptor, and have substantially reduced affinity to the a-subunit of the IL-2 receptor and the high- affinity IL-2 receptor compared to a wild-type form of the IL-2 polypeptide.

Other characteristics of useful mutant IL-2 polypeptides may include the ability to induce proliferation of IL-2 receptor-bearing T and/or NK cells, the ability to induce IL-2 signaling in IL-2 receptor-bearing T and/or NK cells, the ability to generate interferon (IFN)-y as a secondary cytokine by NK cells, a reduced ability to induce elaboration of secondary cytokines - particularly IL-10 and TNF-a - by peripheral blood mononuclear cells (PBMCs), a reduced ability to activate regulatory T cells, a reduced ability to induce apoptosis in T cells, and a reduced toxicity profile in vivo.

Particular mutant IL-2 polypeptides useful in the invention include three amino acid mutations that abolish or reduce affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor. In one aspect, the three amino acid mutations are at positions corresponding to amino acid residues 42, 45, and 72 of a wild-type (WT) human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). In one aspect, the three amino acid mutations are amino acid substitutions. In one aspect, the three amino acid mutations are amino acid substitutions selected from the group of F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, Y45K, L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. In a specific aspect, the three amino acid mutations are amino acid substitutions F42A, Y45A, and L72G (numbering relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147).

In certain aspects, the amino acid mutation reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor by at least 5-fold, specifically at least 10-fold, more specifically at least 25-fold. In certain aspects, where there is more than one amino acid mutation that reduces the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor, the combination of these amino acid mutations may reduce the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL- 2 receptor by at least 30-fold, at least 50-fold, or even at least 10O-fold. In one aspect, the amino acid mutation or combination of amino acid mutations abolishes the affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor, so that no binding is detectable by surface plasmon resonance.

Substantially similar binding to the intermediate-affinity receptor, i.e., preservation of the affinity of the mutant IL-2 polypeptide to said receptor, is achieved when the mutant IL-2 polypeptide exhibits greater than about 70% of the affinity of a wild-type form of the mutant IL-2 polypeptide to the intermediate-affinity IL-2 receptor. Mutant IL-2 polypeptides described herein may exhibit greater than about 80% and even greater than about 90% of such affinity.

Reduction of the affinity of IL-2 polypeptide for the a-subunit of the IL-2 receptor in combination with elimination of the O-glycosylation of IL-2 results in an IL-2 protein with improved properties. For example, elimination of the O-glycosylation site results in a more homogenous product when the mutant IL-2 polypeptide is expressed in mammalian cells such as CHO or HEK cells.

Thus, in certain aspects the mutant IL-2 polypeptide comprises an additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to residue 3 of human IL-2. In one aspect, the additional amino acid mutation which eliminates the O-glycosylation site of IL-2 at a position corresponding to amino acid residue 3 of human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147) is an amino acid substitution. Exemplary amino acid substitutions include T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In a specific aspect, the additional amino acid mutation is the amino acid substitution T3A.

In certain aspects, the mutant IL-2 polypeptide is essentially a full-length IL-2 polypeptide. In certain aspects, the mutant IL-2 polypeptide is a mutant human IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 147 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide including the amino acid sequence of SEQ ID NO: 147 without the mutation. In another aspect, the mutant IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 148 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserve affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 148 without the mutation. In another aspect, the mutant IL-2 polypeptide includes the amino acid sequence of SEQ ID NO: 199 with at least one amino acid mutation that abolishes or reduces affinity of the mutant IL-2 polypeptide to the a-subunit of the IL-2 receptor but preserves affinity of the mutant IL-2 polypeptide to the intermediate affinity IL-2 receptor, compared to an IL-2 polypeptide comprising SEQ ID NO: 199 without the mutation.

In a specific aspect, the mutant IL-2 polypeptide can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, cytokine secretion by an activated T cell or an NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity.

In one aspect, the mutant IL-2 polypeptide has a reduced ability to induce IL-2 signaling in regulatory T cells, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide induces less activation-induced cell death (AICD) in T cells, compared to a wild-type IL-2 polypeptide. In one aspect, the mutant IL-2 polypeptide has a reduced toxicity profile in vivo, compared to a wild-type IL-2 polypeptide. In one embodiment the mutant IL-2 polypeptide has a prolonged serum half-life, compared to a wild-type IL-2 polypeptide.

A particular mutant IL-2 polypeptide useful in the invention includes four amino acid substitutions at positions corresponding to amino acid residues 3, 42, 45, and 72 of a human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). Specific amino acid substitutions are T3A, F42A, Y45A, and L72G. As demonstrated in WO 2012/107417, said quadruple mutant IL-2 polypeptide exhibits no detectable binding to CD25, reduced ability to induce apoptosis in T cells, reduced ability to induce IL-2 signaling in T_reg cells, and a reduced toxicity profile in vivo. However, this mutant IL-2 polypeptide retains the ability to activate IL-2 signaling in effector cells, to induce proliferation of effector cells, and to generate IFN-y as a secondary cytokine by NK cells.

Moreover, the quadruple mutant IL-2 polypeptide has further advantageous properties, such as reduced surface hydrophobicity, good stability, and good expression yield, as described in WO 2012/107417. Unexpectedly, the quadruple mutant IL-2 polypeptide also provides a prolonged serum half-life, compared to wild-type IL-2.

Mutant IL-2 polypeptides useful in the invention, in addition to having mutations in the region of the IL-2 polypeptide that forms the interface of the IL-2 polypeptide with CD25 or the glycosylation site, also may have one or more mutations in the amino acid sequence outside these regions. Such additional mutations in human IL-2 polypeptide may provide additional advantages such as increased expression or stability. For example, the cysteine at position 125 (numbered relative to the human IL- 2 polypeptide sequence of SEQ ID NO: 147) may be replaced with a neutral amino acid such as serine, alanine, threonine or valine, yielding C125S mutant IL-2 polypeptide, C125A mutant IL-2 polypeptide, C125T mutant IL-2 polypeptide, or C125V mutant IL-2 polypeptide, respectively, as described in U.S. Patent No. 4,518,584, which is incorporated herein by reference in its entirety. As described therein, the N-terminal alanine residue of the IL-2 polypeptide may be deleted, yielding such mutants as des-A1 C125S or des-A1 C125A. Alternatively or conjunctively, the mutant IL-2 polypeptide may include a mutation whereby methionine normally occurring at position 104 of a wildtype human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147) is replaced by a neutral amino acid such as alanine (see U.S. Patent No. 5,206,344). The resulting mutants, e. g., des-A1 M104A IL-2, des-A1 M104A C125S IL-2, M104A IL-2, M104A C125A IL-2, des-A1 M104A C125A IL-2, or M104A C125S IL-2 (these and other mutants may be found in U.S. Patent No. 5,1 16,943 and in Weiger et al., Eur J Biochem 180, 295-300 (1989)) may be used in conjunction with the particular IL-2 mutations of the invention.

Thus, in certain aspects, the mutant IL-2 polypeptide includes an additional amino acid mutation at a position corresponding to residue 125 of human IL-2 polypeptide (numbered relative to the human IL-2 polypeptide sequence of SEQ ID NO: 147). In one aspect, the additional amino acid mutation is the amino acid substitution C125A.

The IL-2 mutant may comprise in addition an amino acid substitution at position corresponding to 126 of human IL-2 (shown in SEQ ID NO:147), specifically the amino acid substitution Q126T. The Q126T substitution reduces binding to intermediate-affinity IL-2 receptor (consisting of the p- and y-subunits of the IL-2 receptor), further reducing peripheral activity for the PD-1 -reg-IL-2v molecules in vivo. In one aspect, the mutant IL-2 polypeptide comprises no more than 12, no more than 11 , no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, or no more than 5 amino acid mutations as compared to the corresponding wild-type human IL-2 polypeptide sequence, e.g., the human IL-2 sequence of SEQ ID NO: 147. In a particular aspect, the mutant IL-2 polypeptide comprises no more than 5 amino acid mutations as compared to the corresponding wildtype human IL-2 polypeptide sequence, e.g., the human IL-2 sequence of SEQ ID NO: 147. In another particular aspect, the mutant IL-2 polypeptide comprises no more than 6 amino acid mutations as compared to the corresponding wild-type human IL-2 polypeptide sequence, e.g., the human IL-2 sequence of SEQ ID NO: 147.

In one aspect, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 146. In one aspect, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 146. In one aspect, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 199. In one aspect, the mutant IL-2 polypeptide consists of the sequence of SEQ ID NO: 199.

D. Linkers

Linkers, e.g., peptide linkers, included in the immunoconjugates described herein, may be used to join or connect two polypeptides described herein together. In some aspects, linkers described herein are used to connect an IL-2 polypeptide {e.g., a human IL-2 polypeptide, e.g., a mutant IL-2 polypeptide; e.g., a mutant human IL-2 polypeptide) with a DBA moiety described herein. In certain aspects, the linker is used to connect the IL-2 polypeptide to the DBA moiety, wherein the IL-2 polypeptide is connected to the VH of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus. In certain aspects, the linker is used to connect the IL-2 polypeptide to the DBA moiety, wherein the IL-2 polypeptide is connected to the VL of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus.

In other aspects, a linker may be used to connect an anti-PD-1 antibody moiety or an Fc subunit with an IL-2 polypeptide.

In some aspects, the linker is between 5 to 30 amino acids in length, preferably 20 amino acids in length. In some aspects, the linker comprises the amino acid sequence (G2SG2)x, wherein x is an integer between 1 and 6 (SEQ ID NOs: 149-154). In some aspects, the linker comprises the amino acid sequence of any one of SEQ ID NOs: 149-154). In preferred aspects, the linker comprises the amino acid sequence GGSGGGGSGG (SEQ ID NO: 150) or GGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 152). In preferred aspects, the linker consists of the amino acid sequence GGSGGGGSGG (SEQ ID NO: 150) or GGSGGGGSGGGGSGGGGSGG (SEQ ID NO: 152).

In other aspects, the linker comprises the amino acid sequence (G4S)x, where x is an integer between 1 and 4 (SEQ ID NOs: 192-195). In certain other aspects, the linker comprises the amino acid sequence of SEQ ID NO: 194. In certain other aspects, the linker consists of the amino acid sequence of SEQ ID NO: 194.

E. Immunoconjugates

In some aspects, the immunoconjugates described herein include (a) a first binding domain including: (i) an IL-2 polypeptide described herein; (ii) a linker described herein; and (iii) a dual binding antibody (DBA) moiety described herein that binds to PD-1 and the IL-2 polypeptide in a substantially mutually exclusive manner, wherein the first binding domain is configured such that: (i) when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor; and (ii) when the DBA is bound to PD-1 , the DBA moiety is substantially blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to an IL-2 receptor; and (b) a second binding domain including an anti-PD-1 antibody moiety described herein including a VH and a VL.

For example, in some aspects, the immunoconjugates described herein include (a) a first binding domain including: (i) an IL-2 polypeptide described herein; (ii) a linker described herein; and (iii) a dual binding antibody (DBA) moiety described herein that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus, and wherein the first binding domain is configured such that: (i) when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor; and (ii) when the DBA is bound to PD-1 , the DBA moiety is substantially blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to an IL-2 receptor; and (b) a second binding domain including an anti-PD-1 antibody moiety described herein including a VH and a VL.

In some aspects, the immunoconjugate further includes an Fc domain including a first subunit and a second subunit. In some aspects, the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus; and/or (b) the anti-PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

In some aspects, the Fc domain is an IgG Fc domain. In some aspects, the IgG Fc domain is an IgG 1 Fc domain. In some aspects, the Fc domain is a human IgG Fc domain. In some aspects, the Fc domain is a human IgG 1 Fc domain. In some aspects, the first subunit includes one or more CH domains selected from a first CH2 (CH2i) domain and/or a first CH3 (CH3i) domain; and the second subunit includes one or more CH domains selected from a second CH2 (CH22) domain and/or a second CH3 (CH32) domain. In some aspects, at least one of the one or more CH domains is paired with another CH domain. In some aspects, the CH3i and CH32 domains each include a protuberance or cavity, and wherein the protuberance or cavity in the CH3i domain is positionable in the cavity or protuberance, respectively, in the CH32 domain. In some aspects, the CH3i and CH32 domains meet at an interface between the protuberance and cavity. In some aspects, the CH2i and CH22 domains each include a protuberance or cavity, and wherein the protuberance or cavity in the CH2i domain is positionable in the cavity or protuberance, respectively, in the CH22 domain. In some aspects, the CH2i and CH22 domains meet at an interface between the protuberance and cavity. In some aspects, (a) the first subunit includes a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (b) the first subunit includes a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index).

In some aspects, the first subunit and/or the second subunit includes an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index). In some aspects, the first subunit and the second subunit includes an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index).

In some aspects, the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1 . In some aspects, the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1 .

In some aspects, binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and/or binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 or binding of the anti-PD- 1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 . In some aspects, (a) binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 ; or (b) binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD- 1 to PD-L1 . In some aspects, binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD- L1.

Immunoconjugates described herein may include different combinations of DBA moieties, anti-PD-1 antibody moieties, IL-2 polypeptides, and linkers described herein.

In some aspects, the immunoconjugate may include any DBA moiety as disclosed herein, e.g., in Section A above. In some examples, the DBA moiety includes at least one, at least two, at least three, at least four, at least five, or all six CDRs {e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1. In some instances, the DBA moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1 . In some aspects, the immunoconjugate may include any anti-PD-1 antibody moiety as disclosed herein, e.g., in Section B above. In some examples, the anti-PD-1 antibody moiety includes at least one, at least two, at least three, at least four, at least five, or all six CDRs {e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2. In some instances, the anti-PD-1 antibody moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2.

In some aspects, the immunoconjugate may include any IL-2 moiety as disclosed herein, e.g., in Section C above.

In some aspects, the immunoconjugate may include any linker as disclosed herein, e.g., in Section D above.

Particular immunoconjugates containing particular combinations of DBA moieties, anti-PD-1 antibody moieties, IL-2 polypeptides, and linkers described herein are shown below in Table 3 (containing immunoconjugates having a conventional Fab DBA moiety and a crossFab anti-PD-1 antibody moiety), Table 4 (containing immunoconjugates having a crossFab DBA moiety and a conventional Fab anti-PD-1 antibody moiety), and Table 5 (containing immunoconjugates having a conventional Fab DBA moiety and an scFv anti-PD-1 antibody moiety).

Table 3. Immunoconjugates Having a Conventional Fab DBA moiety and a CrossFab Anti-PD-1 Antibody Moiety.

DBA: DBA moiety; PD-1 : anti-PD-1 antibody; LC: light chain; HC: heavy chain; di: de-immunized; di2: de-immunized2

Table 4. Immunoconjugates Having a Cross Fab DBA moiety and a Conventional Fab Anti-PD-1 Antibody Moiety.

DBA: DBA moiety; PD-1 : anti-PD-1 antibody; LC: light chain; HC: heavy chain; di: de-immunized; opt: affinity-matured

Table 5. Immunoconjugates Having a Conventional Fab DBA moiety and an scFv Anti-PD-1 Antibody Moiety.

DBA: DBA moiety; PD-1 : anti-PD-1 antibody; LC: light chain; HC: heavy chain; di: de-immunized; opt: affinity-matured

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the DBA moiety is AB002345 and the anti-PD-1 antibody moiety is 0376 de-immunized. In some aspects, the immunoconjugate has the construct ID of P1AI7476, P1AI7440, P1AJ1837, P1AI7464, P1 AL2287, P1 AM2983, or P1 AK3171.

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 76; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the DBA moiety is AB003637 and the anti-PD-1 antibody moiety is 0376 de-immunized. In some aspects, the immunoconjugate has the construct ID of P1AI7474, P1AI7438, P1AJ1839, P1AI7462, or P1AK3173.

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti- PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the DBA moiety is AB003637 de-immunized and the anti-PD-1 antibody moiety is 0376 de-immunized. In some aspects, the immunoconjugate has the construct ID of P1 AJ2534 or P1AK3185.

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22. In some aspects, the DBA moiety is AB003637 de-immunized2 and the anti-PD-1 antibody moiety is 0376 de-immunized. In some aspects, the immunoconjugate has the construct ID of P1 AJ2535.

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR-H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3). In some aspects, (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 13 and a VL comprising the amino acid sequence of SEQ ID NO: 4. In some aspects, the DBA moiety is AB002345 and the anti-PD-1 antibody moiety is AB003058. In some aspects, the immunoconjugate has the construct ID of P1AI7473, P1AI7443, P1AI7467, or P1 AK3169.

In some aspects, the immunoconjugate comprises a DBA moiety and an anti-PD-1 antibody moiety wherein: (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39). In some aspects, (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40. In some aspects, (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 40. In some aspects, the DBA moiety is AB002345 and the anti-PD-1 antibody moiety is 1040 affinity-matured. In some aspects, the immunoconjugate has the construct ID of P1 AI7441 , P1 AJ1838, P1 AI7455, or P1 AI7465.

In some aspects, the immunoconjugate includes: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and (iii) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti- PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein: (a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or (b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule.

In some aspects, the immunoconjugate includes: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and (Hi) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is an scFv comprising a VH and a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

In some aspects, the immunoconjugate includes: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (Hi) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL- 2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein: (a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or (b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule.

In some aspects, the immunoconjugate includes: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (Hi) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is an scFv comprising a VH and a VL; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 98. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 98. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 96, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 100, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 98. In some aspects, the immunoconjugate is P1 AI7473.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 102, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 104, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 102, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AI7474.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 96, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AI7476.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 104, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 110, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AJ2534.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 112, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 110, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AJ2535.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 116, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 114, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 116, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 114, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some aspects, the immunoconjugate is P1 AI7438.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some aspects, the immunoconjugate is P1 AI7440.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 128. In some aspects, the immunoconjugate is P1 AI7441 .

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 132. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 122, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 130, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 132. In some aspects, the immunoconjugate is P1 AI7443.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 134, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some aspects, the immunoconjugate is P1 AJ1837. In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 124, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 134, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 128. In some aspects, the immunoconjugate is P1 AJ1838.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 138, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 116, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 138, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 116, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 138, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 116, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 120. In some aspects, the immunoconjugate is P1 AJ1839.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 197, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AL2287.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 198, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 94, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 108. In some aspects, the immunoconjugate is P1 AM2983.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 187, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 185, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 191. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 187, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 185, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 191. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 187, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 185, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 189, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 191. In some aspects, the immunoconjugate is P1 AI7455.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 77, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141 . In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 77, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141 . In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 77, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 141 . In some aspects, the immunoconjugate is P1 AI7462.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 141 . In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 141 . In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 141 . In some aspects, the immunoconjugate is P1 AI7464.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 142. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 142. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 142. In some aspects, the immunoconjugate is P1 AI7465.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 143. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 143. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 143. In some aspects, the immunoconjugate is P1 AI7467.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 144. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 144. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 144. In some aspects, the immunoconjugate is P1 AK3169.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 59, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 59, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some aspects, the immunoconjugate is P1 AK3171.

In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 77, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 77, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 77, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some aspects, the immunoconjugate is P1 AK3173. In one aspect, the immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 92, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 140, and a third polypeptide comprising the amino acid sequence of SEQ ID NO: 145. In one aspect, the immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 92, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 140, and a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 145. In some aspects, the immunoconjugate is P1 AK3185.

In some aspects, the immunoconjugate is a reference immunoconjugate. In some aspects, the reference immunoconjugate includes: (a) a first anti-PD-1 antibody moiety and a second anti-PD-1 antibody moiety, wherein the first and second anti-PD-1 antibody moieties are each a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and (b) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the first anti-PD-1 antibody moiety and the first subunit are connected in the following orientation: N-[first anti-PD-1 antibody moiety]-[first subunit]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, wherein the second anti-PD-1 antibody moiety and the second subunit are connected according to N-[second anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein: (a) the first and second anti-PD-1 antibody moieties are each a conventional Fab molecule, and wherein a mutant IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146, a linker comprising the amino acid sequence of SEQ ID NO: 194, and the first subunit are connected according to N-[first subunit]-[linker]-[mutant IL-2 polypeptide]-C.

In some aspects, each anti-PD-1 antibody moiety of the reference immunoconjugate includes: a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 166), a CDR-H2 comprising the amino acid sequence of TISGGGRDIYYPDSVKG (SEQ ID NO: 167), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 168), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 157), a CDR-L2 comprising the amino acid sequence of RSSTLES (SEQ ID NO: 158), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 159). In some aspects, each anti-PD-1 antibody moiety of the reference immunoconjugate comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 169 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 160. In some aspects, each anti- PD-1 antibody moiety of the reference immunoconjugate comprises a VH comprising the amino acid sequence of SEQ ID NO: 169 and a VL comprising the amino acid sequence of SEQ ID NO: 160. In some aspects, each anti-PD-1 antibody moiety of the reference immunoconjugate is 0376.

In one aspect, the reference immunoconjugate includes a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 170, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 179, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 161 . In one aspect, the reference immunoconjugate includes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 170, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 179, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 161. In one aspect, the reference immunoconjugate includes a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 170, a second polypeptide consisting of the amino acid sequence of SEQ ID NO: 179, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 161. In some aspects, the reference immunoconjugate is P1 AE4422- 14542 (i.e., always-on or always-on P1 AE4422-14542 control).

In some aspects, the reference immunoconjugate is an always on immunoconjugate. In some aspects, the reference immunoconjugate does not include a DBA moiety.

In some aspects, the reference immunoconjugate comprises two DBA moieties. In some aspects, the reference immunoconjugate includes: (a) a first binding domain comprising: (i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 147; (ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (iii) a first DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the first DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the first DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[first DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the first DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the first DBA moiety is bound to PD-1 , the first DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising a second DBA moiety, wherein the second DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VLand wherein when the second DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the second DBA moiety is bound to PD-1 , the second DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; and (c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or (ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the first DBA moiety and the first subunit are connected in the following orientation: N-[first DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the second DBA moiety and the second subunit are connected according to N-[ second DBA moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the first DBA moiety and the second DBA moiety are each a conventional Fab molecule.

In one aspect, the reference immunoconjugate comprises the construct ID P1 AM2158, comprises two DBA moieties having the sequence AB002345, comprises two DBA LCs comprising D1 AW0211 (SEQ ID NO: 59), a DBA-IL-2 HC D1 AN8689 (SEQ ID NO: 136), and a DBA HC C2184344765 (SEQ ID NO: 196).

In some aspects, the reference immunoconjugate includes two DBA moieties wherein: each of the DBA moieties comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). In some aspects, each of the DBA moieties comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58. In some aspects, each of the DBA moieties comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58. In some aspects, each of the DBA moieties is AB002345. In some aspects, the reference immunoconjugate has the construct ID of P1 AM2158 (i.e., Compound C).

In one aspect, the reference immunoconjugate includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 136, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 196. In one aspect, the immunoconjugate includes a first polypeptide and a polypeptide each comprising the amino acid sequence of SEQ ID NO: 59, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 136, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 196. In one aspect, the immunoconjugate includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 59, a third polypeptide consisting of the amino acid sequence of SEQ ID NO: 136, and a fourth polypeptide consisting of the amino acid sequence of SEQ ID NO: 196. In some aspects, the reference immunoconjugate is P1AM2158 (Compound C).

In some aspects, the invention provides murine analogue versions of the immunoconjugates provided herein. Methods of making murine analogues are known in the art. Construct IDs for murine analogues of immunoconjugates disclosed herein are shown below in Table 6.

Table 6. Murine Analogues of Human Immunoconjugate Constructs.

DBA: DBA moiety; PD-1 : anti-PD-1 antibody; LC: light chain; HC: heavy chain; di: de-immunized; opt: affinity-matured

F. Additional Antibodies

In some aspects, any of the additional antibodies described in this section can be used in the context of an immunoconjugate (e.g., in a PD-1 -regulated IL-2 immunoconjugate) described herein. In some aspects, an immunoconjugate (e.g., in a PD-1 -regulated IL-2 immunoconjugate) described herein may comprise any of the additional antibodies described in this section, e.g., comprising one or more of any of the additional antibodies described in this section, any one or more of the linkers described herein (e.g., as described in Section D) and/or any one or more of the IL-2 polypeptides described herein (e.g., as described in Section C). In some aspects, the IL-2 polypeptide(s) may be linked to the N- or C- terminus of any of the heavy and/or light chains of the additional antibody, e.g., via a linker, to generate an immunoconjugate. In a preferred aspect, the IL-2 polypeptide(s) may be linked to the N- terminus of any of the heavy and/or light chains of the additional antibody, e.g., via a linker, to generate an immunoconjugate described herein.

In some aspects, an antibody includes two DBA moieties each comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57). In some aspects, each DBA moiety of the antibody is AB002345.

In some aspects, each DBA moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 69; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 70; (Hi) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 71 ; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 72; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 60; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 61 ; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 62; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 63. In some aspects, each DBA moiety of the antibody comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 67; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 58; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 67; (b) a VL comprising the amino acid sequence of SEQ ID NO: 58; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody is AB002345.

In some aspects, an antibody includes two DBA moieties each comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75). In some aspects, each DBA moiety of the antibody is AB003637 or AB003637 de-immunized.

In some aspects, each DBA moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 87; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (Hi) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 89; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 90; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 80; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 81 . In some aspects, each DBA moiety of the antibody comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 76; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising the amino acid sequence of SEQ ID NO: 76; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody is AB003637.

In some aspects, each DBA moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 87; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 88; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 89; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 90; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 78; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 79; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 80; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 155. In some aspects, the DBA moiety comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 85; (b) a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or (c) a VH as in (a) and a VL as in (b). In some aspects, each DBA moiety of the antibody is AB003637 de-immunized.

In some aspects, an antibody includes two anti-PD-1 antibody moieties each comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR- H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3). In some aspects, each anti-PD-1 antibody moiety of the antibody is AB003058.

In some aspects, each anti-PD-1 antibody moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 15; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 16; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 17; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 18; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 6; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 7; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 8; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 9. In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 13; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 13; (b) a VL comprising the amino acid sequence of SEQ ID NO: 4; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody is AB003058.

In some aspects, an antibody includes two anti-PD-1 antibody moieties each comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR- H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ). In some aspects, each anti-PD-1 antibody moiety of the antibody is 0376 de-immunized.

In some aspects, each anti-PD-1 antibody moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 33; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 34; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 35; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 36; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 24; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 25; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 26; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 27. In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31 ; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 22; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 31 ; (b) a VL comprising the amino acid sequence of SEQ ID NO: 22; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody is 0376 de-immunized.

In some aspects, an antibody includes two anti-PD-1 antibody moieties each comprising the following six CDRs: a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR- H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39). In some aspects, each anti-PD-1 antibody moiety of the antibody is 1040 affinity-matured.

In some aspects, each anti-PD-1 antibody moiety of the antibody comprises one or more of the following eight framework regions (FRs): (i) an FR-H1 comprising the amino acid sequence of SEQ ID NO: 51 ; (ii) an FR-H2 comprising the amino acid sequence of SEQ ID NO: 52; (iii) an FR-H3 comprising the amino acid sequence of SEQ ID NO: 53; (iv) an FR-H4 comprising the amino acid sequence of SEQ ID NO: 54; (v) an FR-L1 comprising the amino acid sequence of SEQ ID NO: 42; (vi) an FR-L2 comprising the amino acid sequence of SEQ ID NO: 43; (vii) an FR-L3 comprising the amino acid sequence of SEQ ID NO: 44; and/or (viii) an FR-L4 comprising the amino acid sequence of SEQ ID NO: 45. In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 49; (b) a VL comprising an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 40; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody comprises: (a) a VH comprising the amino acid sequence of SEQ ID NO: 49; (b) a VL comprising the amino acid sequence of SEQ ID NO: 40; or (c) a VH as in (a) and a VL as in (b). In some aspects, each anti-PD-1 antibody moiety of the antibody is 1040 affinity-matured.

In some aspects, the antibody further includes an Fc domain including a first subunit and a second subunit. In some aspects, a first anti-PD-1 antibody moiety and the first subunit are connected in the following orientation: N-[ first anti-PD-1 antibody moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus; and/or (b) a second anti- PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[second anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

In some aspects, the Fc domain is an IgG Fc domain. In some aspects, the IgG Fc domain is an IgGi Fc domain.

In some aspects, the first subunit and/or the second subunit includes an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index).

In some aspects, the antibodies may include any DBA moiety as disclosed herein, e.g., in Section A above. In some examples, the DBA moiety includes at least one, at least two, at least three, at least four, at least five, or all six CDRs {e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1. In some instances, the DBA moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 1 .

In some aspects, the antibodies may include any anti-PD-1 antibody moiety as disclosed herein, e.g., in Section B above. In some examples, the anti-PD-1 antibody moiety includes at least one, at least two, at least three, at least four, at least five, or all six CDRs {e.g., comprises one, two, three, four, five, or six CDRs) comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2. In some instances, the anti-PD-1 antibody moiety comprises a VH and/or a VL comprising amino acid sequence(s) of the SEQ ID NOs as illustrated in Table 2.

Antibodies described herein may include different DBA moieties or anti-PD-1 antibody moieties. Particular antibodies containing particular combinations of DBA moieties or anti-PD-1 antibody moieties described herein are shown below in Table 7.

Table 7. Antibodies (contains two of each HC and LC)

DBA: DBA moiety; PD-1 : anti-PD-1 antibody; LC: light chain; HC: heavy chain; di: de-immunized; di2: de-immunized2

In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 132. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 14, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 132. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 14, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 132. In some aspects, the antibody is P1 AI7512.

In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 32, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 32, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 120. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 32, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 120. In some aspects, the antibody is P1AH4157.

In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 128. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 50, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 128. In some aspects, the antibody is P1 AG3741 .

In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 59. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 59. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 68, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 59. In some aspects, the antibody is P1 AI3784.

In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 77. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 77. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 77. In some aspects, the antibody is P1AI7516. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 92. In one aspect, the antibody includes a first polypeptide and a second polypeptide each comprising the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each comprising the amino acid sequence of SEQ ID NO: 92. In one aspect, the antibody includes a first polypeptide and a second polypeptide each consisting of the amino acid sequence of SEQ ID NO: 86, and a third polypeptide and a fourth polypeptide each consisting of the amino acid sequence of SEQ ID NO: 92. In some aspects, the antibody is P1 AI7514.

G. Properties of Immunoconjugates and/or Antibodies

In some examples, the immunoconjugates (e.g., PD-1 -regulated IL-2 immunoconjugates) and/or antibodies {e.g., DBAs and/or anti-PD-1 antibodies) disclosed herein may include any of the properties or any combination of the properties set forth in Sections 1 -6 below.

1 . Antibody Fragments

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) described herein includes one or more antibody fragments. In certain aspects, an immunoconjugate (e.g., a PD-1 - regulated IL-2 immunoconjugate) or an antibody provided herein includes an antibody fragment.

Any suitable antibody fragment may be used. In one aspect, the antibody fragment {e.g., a DBA moiety or an anti-PD-1 antibody moiety) is a Fab, Fab’, Fab’-SH, or F(ab’)2 fragment. In particular examples, the antibody fragment {e.g., a DBA moiety or an anti-PD-1 antibody moiety) is a Fab fragment. Papain digestion of intact antibodies produces two identical antigen-binding fragments, called Fab fragments containing each the heavy- and light-chain variable domains (VH and VL, respectively) and also the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1 ). Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab’)2 fragment that has two antigenbinding sites (two Fab fragments) and a part of the Fc region. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

In another aspect, the antibody fragment is a diabody, a triabody, or a tetrabody.

In a further aspect, the antibody fragment is a single chain Fab fragment.

In another aspect, the antibody fragment {e.g., an anti-PD-1 antibody moiety) is single-chain variable fragment (scFv). In another aspect, the antibody fragment is a single-domain antibody.

Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as recombinant production by recombinant host cells {e.g., E. coli), as described herein.

2. Chimeric and Humanized Immunoconjugates and/or Antibodies

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein is a chimeric immunoconjugate or antibody. In certain aspects, an immunoconjugate (e.g., a PD-1 - regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein includes a chimeric immunoconjugate or antibody.

Certain chimeric antibodies are described, e.g., in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851 -6855 (1984)). In one example, a chimeric antibody comprises a non-human variable region {e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain aspects, a chimeric immunoconjugate or antibody is a humanized immunoconjugate or antibody. Typically, a non-human immunoconjugate or antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized immunoconjugate or antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human immunoconjugate or antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized immunoconjugate or antibody optionally will also comprise at least a portion of a human constant region. In some aspects, some FR residues in a humanized immunoconjugate or antibody are substituted with corresponding residues from a non-human immunoconjugate or antibody {e.g., the antibody or antibody fragment(s) from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et a!., Proc. Nat’l Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5, 821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991 ) (describing “resurfacing”); Dall’Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61 -68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :22611 - 22618 (1996)).

3. Human Immunoconjugates and Antibodies

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein is or includes a human immunoconjugate or antibody. Human immunoconjugates or antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001 ) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal’s chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; U.S. Patent No. 5,770,429 describing HUMAB® technology; U.S. Patent No. 7,041 ,870 describing K-M MOUSE® technology, and U.S. Patent Application Publication No. US 2007/0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991 ).) Human antibodies generated via human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265- 268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.

4. Multispecific Immunoconjugates and Multispecific Antibodies

In certain aspects, a multispecific immunoconjugate or a multispecific antibody is disclosed herein. “Multispecific immunoconjugates” are monoclonal immunoconjugates that have binding specificities for at least two different sites, i.e., different epitopes on different antigens or different epitopes on the same antigen. In certain aspects, the multispecific immunoconjugate or multispecific antibody has three or more binding specificities. In certain aspects, one of the binding specificities is for an IL-2 polypeptide or PD-1 in a mutually exclusive manner and the other specificity is for PD-1 only. Multispecific {e.g., bispecific) immunoconjugates or antibodies may also be used to localize cytotoxic agents {e.g., IL-2 polypeptides) or cells to cells which express PD-1 . Multispecific immunoconjugates or antibodies may be prepared as full-length immunoconjugates or antibodies or antibody fragments.

Techniques for making multispecific immunoconjugates or antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and “knob-in-hole” engineering (see, e.g., U.S. Patent No. 5,731 ,168, and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multi-specific immunoconjugates or antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see, e.g., WO 2009/089004); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan etal., Science, 229: 81 (1985)); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5) :1547-1553 (1992) and WO 2011/034605); using the common light chain technology for circumventing the light chain mis-pairing problem (see, e.g., WO 98/50431 ); using “diabody” technology for making bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodies as described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991 ). 5. Immunoconjugate and Antibody Variants

In certain aspects, amino acid sequence variants of the immunoconjugates, antibodies, or IL- 2 polypeptides provided herein are contemplated. For example, it may be desirable to alter the binding affinity and/or other biological properties of the immunoconjugate or antibody. Amino acid sequence variants of a PD-1 -regulated IL-2 immunoconjugate or antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the immunoconjugate or antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the immunoconjugates, antibodies, or IL-2 polypeptides. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. a) Substitution, Insertion, and Deletion Variants

In certain aspects, immunoconjugates, antibodies, or IL-2 polypeptides having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and FRs.

Conservative substitutions are shown in Table 8 under the heading of “conservative substitutions”. More substantial changes are provided in Table 8 under the heading of “exemplary substitutions”, and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Amino acids may be grouped according to common side-chain properties:

(1 ) hydrophobic: Norleucine, Met, Ala, Vai, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody. Generally, the resulting variant(s) selected for further study will have modifications {e.g., improvements) in certain biological properties {e.g., increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g., using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity {e.g., binding affinity).

Alterations {e.g., substitutions) may be made in CDRs, e.g., to improve antibody affinity. Such alterations may be made in CDR “hotspots”, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues that contact antigen, with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1 -37 (O’Brien et al., ed., Human Press, Totowa, NJ, (2001 ).) In some aspects of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods {e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves CDR-directed approaches, in which several CDR residues {e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding may be specifically identified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations {e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in the CDRs. Such alterations may, for example, be outside of antigen contacting residues in the CDRs. In certain variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called “alanine scanning mutagenesis” as described by Cunningham and Wells (1989) Science, 244:1081 -1085. In this method, a residue or group of target residues {e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid {e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme {e.g., for ADEPT (antibody directed enzyme prodrug therapy)) or a polypeptide which increases the serum half-life of the antibody. b) Glycosylation variants

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein is altered to increase or decrease the extent to which the immunoconjugate or antibody is glycosylated. Addition or deletion of glycosylation sites to a PD-1 -regulated IL-2 immunoconjugate or an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

Where the immunoconjugate or antibody comprises an Fc region, the oligosaccharide attached thereto may be altered. Native immunoconjugates or antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N- linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GIcNAc), galactose, and sialic acid, as well as a fucose attached to a GIcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody as described herein may be made in order to create antibody variants with certain improved properties.

In one aspect, antibody variants are provided having a non-fucosylated oligosaccharide, i.e., an oligosaccharide structure that lacks fucose attached (directly or indirectly) to an Fc region. Such non-fucosylated oligosaccharide (also referred to as “afucosylated” oligosaccharide) particularly is an N-linked oligosaccharide which lacks a fucose residue attached to the first GIcNAc in the stem of the biantennary oligosaccharide structure, and such antibodies are further referred to herein as an “afucosylated antibodies.” In one aspect, antibody variants are provided having an increased proportion of non-fucosylated oligosaccharides in the Fc region as compared to a native or parent antibody. For example, the proportion of non-fucosylated oligosaccharides may be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucosylated oligosaccharides are present). In certain embodiments, the proportion of afucosylation is between about 65% to about 100%, between about 80% to about 100%, or between about 80% to about 95%. The percentage of non-fucosylated oligosaccharides is the (average) amount of oligosaccharides lacking fucose residues, relative to the sum of all oligosaccharides attached to Asn 297 (e. g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2006/082515, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (EU numbering of Fc region residues); however, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies, e.g., Asn 299. Such antibodies having an increased proportion of non-fucosylated oligosaccharides in the Fc region may have improved FcyRllla receptor binding and/or improved effector function, in particular improved ADCC function. See, e.g., US 2003/0157108; US 2004/0093621 .

In one aspect, the present disclosure provides afucosylated antibody variants that have enhanced FcyRllla receptor binding. In one aspect, the present disclosure provides afucosylated antibody variants that have enhanced antibody-dependent cellular cytotoxicity (ADCC). In one aspect, the present disclosure provides afucosylated antibody variants that have antibody-dependent cellular phagocytosis (ADCP) activities.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Led 3 CHO cells deficient in protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, especially at Example 11 ), and knockout cell lines, such as alpha-1 ,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107), or cells with reduced or abolished activity of a GDP-fucose synthesis or transporter protein (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282). See also Pereira et al., MABS (2018) 693-711 .

In a further aspect, antibody variants are provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GIcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function as described above. Examples of such antibody variants are described, e.g., in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851 -861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764. c) Fc region variants

In certain aspects, one or more amino acid modifications may be introduced into the Fc region of a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti- PD-1 antibody moieties) provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence {e.g., a human IgGi, lgG2, IgGs or lgG4 Fc region) comprising an amino acid modification {e.g., a substitution) at one or more amino acid positions.

In certain aspects, the invention contemplates a PD-1 -regulated IL-2 immunoconjugate or antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 ). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat’l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499-1502 (1985); 5,821 ,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351 -1361 (1987)). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA; and CYTOTOX 96® non-radioactive cytotoxicity assay (Promega, Madison, Wl). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat’l Acad. Sci. USA 95:652-656 (1998). C1 q binding assays may also be carried out to confirm that the antibody is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101 :1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int’l. Immunol. 18(12):1759-1769 (2006); WO 2013/120929 Al).

Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called “DANA” Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581 ).

Certain immunoconjugate or antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312, and Shields etal., J. Biol. Chem. 9(2): 6591 -6604 (2001 ).)

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) or antibody variant comprises an Fc region with one or more amino acid substitutions which improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) or antibody variant comprises an Fc region with one or more amino acid substitutions which diminish FcyR binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In one aspect, the substitutions are L234A and L235A (LALA). In certain aspects, the immunoconjugate or antibody variant further comprises D265A and/or P329G in an Fc region (EU numbering of residues) derived from a human IgG 1 Fc region. In one aspect, the substitutions are L234A, L235A and P329G (LALA- PG) in an Fc region derived from a human IgGi Fc region. (See, e.g., WO 2012/130831 ). In another aspect, the substitutions are L234A, L235A and D265A (LALA-DA) in an Fc region derived from a human IgG 1 Fc region. In certain aspects, the Fc region comprises a modification configured to promote the association of the first Fc subunit with the second Fc subunit. “Knob-in-hole” engineering of immunoconjugate or antibodies may be utilized to generate a first arm containing a knob and a second arm containing the hole into which the knob of the first arm may bind. The knob of the multispecific antibodies of the invention may include a DBA moiety in one embodiment. Alternatively, the knob of the immunoconjugates or antibodies of the invention may include an anti-PD-1 antibody moiety. The hole of the immunoconjugates or antibodies of the invention may include a DBA moiety. Alternatively, the hole of the immunoconjugates or antibodies of the invention may include an anti-PD- 1 antibody moiety. Immunoconjugates and antibodies may also be engineered using immunoglobulin crossover (also known as Fab domain exchange or CrossMab format) technology (see e.g., WO 2009/080253; Schaefer et al., Proc. Natl. Acad. Sci. USA, 108:11187-11192 (2011 )). Immunoconjugates or antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (WO 2009/089004A1 ); cross-linking two or more antibodies or fragments (see, e.g., US Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); or by using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)).

An amino acid residue in the CH3 domain of the second Fc subunit may be replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance {e.g., a knob) within the CH3 domain of the second Fc subunit which is positionable in a cavity {e.g., a hole) within the CH3 domain of the first Fc subunit, and an amino acid residue in the CH3 domain of the first Fc subunit may be replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity {e.g., a hole) within the CH3 domain of the first Fc subunit within which the protuberance {e.g., a knob) within the CH3 domain of the second Fc subunit may be positionable. In some embodiments, the CH3 domain of the second Fc subunit comprises the amino acid substitution of T366, and the CH3 domain of the first Fc subunit comprises amino acid substitutions at one, two, or all three of T366, L368, and/or Y407. In some embodiments, the CH3 domain of the second Fc subunit comprises the amino acid substitution of T366W, and the CH3 domain of the first Fc subunit comprises one, two, or all three amino acid substitutions of T366S, L368A, and/or Y407V.

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) comprises an Fc region with one or more amino acid substitutions which improve FcyR binding (and thereby improve effector function), e.g., substitutions at positions. In certain aspects, the antibody variant comprises an Fc region with at least one amino acid substitutions of G236A, I332E, S298A, E333A, K334A, S239D, A330L, F243L, R292P, Y300L, V305I, P396L, L235V, L234Y, L235Q, G236W, S239M, H268D, D270E, K326D, A330M, K334E (EU numbering of residues) (See, e.g., Liu et al., Antibodies (Basel) (2020);9(4):64). In some aspects, alterations are made in the Fc region that result in altered (/.e., either improved or diminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in US Patent No. 6,194,551 , WO 99/51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

Antibodies with increased half lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.). Those antibodies comprise an Fc region with one or more substitutions therein which improve binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more of Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434 (EU numbering of residues), e.g., substitution of Fc region residue 434 (See, e.g., US Patent No. 7,371 ,826; Dall’Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524).

Fc region residues critical to the mouse Fc-mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g., Dall’Acqua, W.F., et al. J. Immunol 169 (2002) 5171 -5180). Residues I253, H310, H433, N434, and H435 (EU numbering of residues) are involved in the interaction (Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533; Firan, M., et al., Int. Immunol. 13 (2001 ) 993; Kim, J.K., et al., Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn (Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819). Studies of the human Fc-human FcRn complex have shown that residues I253, S254, H435, and Y436 are crucial for the interaction (Firan, M., et al., Int. Immunol. 13 (2001 ) 993; Shields, R.L., et al., J. Biol. Chem. 276 (2001 ) 6591 -6604). In Yeung, Y.A., et al. (J. Immunol. 182 (2009) 7667-7671 ) various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 (EU numbering of residues) have been reported and examined.

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 253, and/or 310, and/or 435 of the Fc-region (EU numbering of residues). In certain aspects, the immunoconjugate or antibody comprises an Fc region with the amino acid substitutions at positions 253, 310 and 435. In one aspect, the substitutions are I253A, H310A and H435A in an Fc region derived from a human lgG1 Fc-region. See, e.g., Grevys, A., et al., J. Immunol. 194 (2015) 5497-5508.

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) comprises an Fc region with one or more amino acid substitutions, which reduce FcRn binding, e.g., substitutions at positions 310, and/or 433, and/or 436 of the Fc region (EU numbering of residues). In certain aspects, the immunoconjugate or antibody comprises an Fc region with the amino acid substitutions at positions 310, 433 and 436. In one aspect, the substitutions are H310A, H433A and Y436A in an Fc region derived from a human lgG1 Fc-region. (See, e.g., WO 2014/177460 Al).

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) comprises an Fc region with one or more amino acid substitutions which increase FcRn binding, e.g., substitutions at positions 252, and/or 254, and/or 256 of the Fc region (EU numbering of residues). In certain aspects, the immunoconjugate or antibody comprises an Fc region with amino acid substitutions at positions 252, 254, and 256. In one aspect, the substitutions are M252Y, S254T and T256E in an Fc region derived from a human IgGi Fc-region. See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821 ; and WO 94/29351 concerning other examples of Fc region variants.

The C-terminus of the heavy chain of the immunoconjugate or antibody as reported herein can be a complete C-terminus ending with the amino acid residues PGK. The C-terminus of the heavy chain can be a shortened C-terminus in which one or two of the C terminal amino acid residues have been removed. In one aspect, the C-terminus of the heavy chain is a shortened C-terminus ending PG. In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain as specified herein, comprises the C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbering of amino acid positions). In one aspect of all aspects as reported herein, a PD-1 -regulated IL-2 immunoconjugate or an antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C-terminal glycine residue (G446, EU index numbering of amino acid positions). In one aspect of all aspects as reported herein, an antibody comprising a heavy chain including a C-terminal CH3 domain, as specified herein, comprises a C- terminal proline residue (P445, EU index numbering of amino acid positions). d) Cysteine Engineered Immunoconjugate and Antibody Variants

In certain aspects, it may be desirable to create cysteine engineered immunoconjugates or antibodies, e.g., THIOMAB™ antibodies, in which one or more residues of an immunoconjugate or an antibody are substituted with cysteine residues. In particular aspects, the substituted residues occur at accessible sites of the immunoconjugate or antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create a PD-1 -regulated IL-2 immunoconjugate, as described further herein. Cysteine engineered immunoconjugates or antibodies may be generated as described, e.g., in U.S. Patent No. 7,521 ,541 , 8,30,930, 7,855,275, 9,000,130, or WO 2016040856. e) Immunoconjugate and Antibody Derivatives

In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the immunoconjugate or antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1 , 3-dioxolane, poly-1 ,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols {e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

6. Fc domain modifications promoting heterodimerization

Immunoconjugates and antibodies of the invention may comprise one or more DBA moieties and/or one or more anti-PD-1 antibody moieties, fused to one or the other of the two subunits of an Fc domain. Thus, the two subunits of the Fc domain are typically comprised in two non-identical polypeptide chains. Recombinant co-expression of these polypeptides and subsequent dimerization can lead to several possible combinations of the two polypeptides. To improve the yield and purity of immunoconjugates and antibodies in recombinant production, in some examples it is advantageous to introduce in the Fc domain of the immunoconjugate or antibody a modification promoting the association of the desired polypeptides.

Accordingly, in particular embodiments, a Fc domain of the immunoconjugate or antibody according to the invention may comprise a modification promoting the association of the first and the second subunit of the Fc domain. The site of most extensive protein-protein interaction between the two subunits of a human IgG Fc domain is in the CH3 domain of the Fc domain. Thus, in one embodiment the modification is in the CH3 domain of the Fc domain. There exist several approaches for modifications in the CH3 domain of the Fc domain in order to enforce heterodimerization, which are described e.g., in WO 96/27011 , WO 98/050431 , EP 1870459, WO 2007/110205, WO 2007/147901 , WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO 2012058768, WO 2013157954, WO 2013096291 . Typically, in such approaches the CH3 domain of the first subunit of the Fc domain and the CH3 domain of the second subunit of the Fc domain are both engineered in a complementary manner so that each CH3 domain (or the heavy chain comprising it) can no longer homodimerize with itself but is forced to heterodimerize with the complementarily engineered other CH3 domain (so that the first and second CH3 domain heterodimerize and no homodimers between the two first or the two second CH3 domains are formed). These different approaches for improved heavy chain heterodimerization are contemplated as different alternatives in combination with the heavy-light chain modifications (VH and VL exchange/replacement in one binding arm and the introduction of substitutions of charged amino acids with opposite charges in the CH1/CL interface) in the immunoconjugate or antibody according to the invention which reduce light chain mispairing and Bence Jones-type side products.

In a specific embodiment, the modification promoting the association of the first and the second subunit of the Fc domain is a so-called “knob-into-hole” modification, comprising a “knob” modification in one of the two subunits of the Fc domain and a “hole” modification in the other one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g., in US 5,731 ,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001 ). Generally, the method involves introducing a protuberance (“knob”) at the interface of a first polypeptide and a corresponding cavity (“hole”) in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains {e.g., tyrosine or tryptophan). Compensatory cavities of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones {e.g., alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the first subunit of the Fc domain of the immunoconjugate or antibody an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the CH3 domain of the first subunit which is positionable in a cavity within the CH3 domain of the second subunit, and in the CH3 domain of the second subunit of the Fc domain an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the CH3 domain of the second subunit within which the protuberance within the CH3 domain of the first subunit is positionable.

Preferably, the amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protuberance and cavity can be made by altering the nucleic acid encoding the polypeptides, e.g., by site-specific mutagenesis, or by peptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of the Fc domain (the “knobs” subunit) the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and in the CH3 domain of the second subunit of the Fc domain (the “hole” subunit) the tyrosine residue at position 407 is replaced with a valine residue (Y407V). In one embodiment, in the second subunit of the Fc domain additionally the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numberings according to Kabat EU index).

In yet a further embodiment, in the first subunit of the Fc domain additionally the serine residue at position 354 is replaced with a cysteine residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine residue (E356C), and in the second subunit of the Fc domain additionally the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) (numberings according to Kabat EU index). Introduction of these two cysteine residues results in formation of a disulfide bridge between the two subunits of the Fc domain, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001 )).

In a particular embodiment, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W, and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T366S, L368A and Y407V (numbering according to Kabat EU index).

Other techniques of CH3-modification for enforcing the heterodimerization are contemplated as alternatives according to the invention and are described e.g., in WO 96/27011 , WO 98/050431 , EP 1870459, WO 2007/110205, WO 2007/147901 , WO 2009/089004, WO 2010/129304, WO 2011 /90754, WO 2011 /143545, WO 2012/058768, WO 2013/157954, WO 2013/096291 .

In one embodiment the heterodimerization approach described in EP 1870459 A1 , is used alternatively. This approach is based on the introduction of charged amino acids with opposite charges at specific amino acid positions in the CH3/CH3 domain interface between the two subunits of the Fc domain. One preferred embodiment for the immunoconjugate or antibody of the invention are amino acid mutations R409D; K370E in one of the two CH3 domains (of the Fc domain) and amino acid mutations D399K; E357K in the other one of the CH3 domains of the Fc domain (numbering according to Kabat EU index).

In another embodiment, the immunoconjugate or antibody of the invention comprises amino acid mutation T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (numberings according to Kabat EU index).

In another embodiment, the immunoconjugate or antibody of the invention comprises amino acid mutations S354C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of the second subunit of the Fc domain, or the immunoconjugate or antibody comprises amino acid mutations Y349C, T366W in the CH3 domain of the first subunit of the Fc domain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3 domains of the second subunit of the Fc domain and additionally amino acid mutations R409D; K370E in the CH3 domain of the first subunit of the Fc domain and amino acid mutations D399K; E357K in the CH3 domain of the second subunit of the Fc domain (all numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO 2013/157953 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutation T366K and a second CH3 domain comprises amino acid mutation L351 D (numberings according to Kabat EU index). In a further embodiment the first CH3 domain comprises further amino acid mutation L351 K. In a further embodiment the second CH3 domain comprises further an amino acid mutation selected from Y349E, Y349D and L368E (preferably L368E) (numberings according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO 2012/058768 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations L351 Y, Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain comprises a further amino acid mutation at position T411 , D399, S400, F405, N390, or K392, e.g. selected from a) T411 N, T411 R, T411 Q, T411 K, T411 D, T411 E or T411 W, b) D399R, D399W, D399Y or D399K, c) S400E, S400D, S400R, or S400K, d) F405I, F405M, F405T, F405S, F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L, K392F, or K392E (numberings according to Kabat EU index). In a further embodiment a first CH3 domain comprises amino acid mutations L351 Y, Y407A and a second CH3 domain comprises amino acid mutations T366V, K409F. In a further embodiment a first CH3 domain comprises amino acid mutation Y407A and a second CH3 domain comprises amino acid mutations T366A, K409F. In a further embodiment the second CH3 domain further comprises amino acid mutations K392E, T411 E, D399R, and S400R (numberings according to Kabat EU index).

In one embodiment, the heterodimerization approach described in WO 2011/143545 is used alternatively, e.g., with the amino acid modification at a position selected from the group consisting of 368 and 409 (numbering according to Kabat EU index).

In one embodiment the heterodimerization approach described in WO 2011/090762, which also uses the knobs-into-holes technology described above, is used alternatively. In one embodiment, a first CH3 domain comprises amino acid mutation T366W and a second CH3 domain comprises amino acid mutation Y407A. In one embodiment, a first CH3 domain comprises amino acid mutation T366Y and a second CH3 domain comprises amino acid mutation Y407T (numberings according to Kabat EU index).

In one embodiment, the immunoconjugate or antibody or its Fc domain is of lgG2 subclass and the heterodimerization approach described in WO 2010/129304 is used alternatively.

In an alternative embodiment, a modification promoting association of the first and the second subunit of the Fc domain comprises a modification mediating electrostatic steering effects, e.g., as described in PCT publication WO 2009/089004. Generally, this method involves replacement of one or more amino acid residues at the interface of the two Fc domain subunits by charged amino acid residues so that homodimer formation becomes electrostatically unfavorable but heterodimerization electrostatically favorable. In one such embodiment, a first CH3 domain comprises amino acid substitution of K392 or N392 with a negatively charged amino acid {e.g., glutamic acid (E), or aspartic acid (D), preferably K392D or N392D) and a second CH3 domain comprises amino acid substitution of D399, E356, D356, or E357 with a positively charged amino acid {e.g., lysine (K) or arginine (R), preferably D399K, E356K, D356K, or E357K, and more preferably D399K and E356K). In a further embodiment, the first CH3 domain further comprises amino acid substitution of K409 or R409 with a negatively charged amino acid {e.g., glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). In a further embodiment the first CH3 domain further or alternatively comprises amino acid substitution of K439 and/or K370 with a negatively charged amino acid {e.g., glutamic acid (E), or aspartic acid (D)) (all numberings according to Kabat EU index).

In yet a further embodiment, the heterodimerization approach described in WO 2007/147901 is used alternatively. In one embodiment a first CH3 domain comprises amino acid mutations K253E, D282K, and K322D and a second CH3 domain comprises amino acid mutations D239K, E240K, and K292D (numberings according to Kabat EU index).

In still another embodiment, the heterodimerization approach described in WO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises amino acid substitutions K392D and K409D, and the second subunit of the Fc domain comprises amino acid substitutions D356K and D399K (numbering according to Kabat EU index).

H. Recombinant Methods and Compositions

An immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) may be produced using recombinant methods and compositions, e.g., as described in US 4,816,567 and in U.S. Publication No. 2013/0078249, each of which is incorporated herein by reference in its entirety. In one embodiment, an isolated nucleic acid {e.g., a polynucleotide) or a set of isolated nucleic acids encoding a PD-1 - regulated IL-2 immunoconjugate or an antibody described herein is provided. In one embodiment, an isolated nucleic acid {e.g., a polynucleotide) or a set of isolated nucleic acids encoding a PD-1 - regulated IL-2 immunoconjugate or an antibody, or fragment thereof, described herein is provided. Such nucleic acid or set of nucleic acids may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising a VH of the immunoconjugate or antibody {e.g., the light and/or heavy chains of the either arm of the immunoconjugate or antibody). In a further embodiment, one or more vectors {e.g., expression vectors) comprising such nucleic acid or set of nucleic acids are provided. Polynucleotides encoding a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) of the invention may be expressed as a single polynucleotide molecule or as multiple {e.g., two or more) polynucleotides that are coexpressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate or antibody. For example, a light chain portion of an antigen binding moiety {e.g., a DBA moiety or an anti-PD-1 antibody moiety) may be encoded by a separate polynucleotide from the portion of the immunoconjugate or antibody comprising the heavy chain portion of the antigen binding moiety, an Fc domain subunit. When coexpressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety. In another example, the portion of the immunoconjugate or antibody comprising one of the two Fc domain subunits could be encoded by a separate polynucleotide from the portion of the immunoconjugate or antibody comprising the other of the two Fc domain subunits. When co-expressed, the Fc domain subunits will associate to form the Fc domain.

In certain embodiments, an isolated polynucleotide of the invention encodes a fragment of a PD-1 -regulated IL-2 immunoconjugate or antibody comprising a first and a second antigen-binding domain, and an Fc domain consisting of two subunits. In one embodiment, an isolated polynucleotide of the invention encodes the heavy chain of a first antigen binding moiety {e.g., a DBA moiety or an anti-PD-1 antibody moiety) and a subunit of the Fc domain. In another embodiment, an isolated polynucleotide of the invention encodes the heavy chain of a second antigen binding moiety and a subunit of the Fc domain. In a more specific embodiment, the isolated polynucleotide encodes a polypeptide, wherein a Fab heavy chain (or scFv) shares a C-terminal peptide bond with an Fc domain subunit. In some embodiments, the light chains of the first and second antigen-binding moieties are co-expressed and associate with the heavy chain regions to form Fab domains.

In a further embodiment, a host cell comprising such nucleic acid or set of nucleic acids is provided. In one such embodiment, a host cell comprises {e.g., has been transformed with): (1 ) a vector comprising a nucleic acid that encodes an amino acid sequence comprising at least one VL of the immunoconjugate or antibody and an amino acid sequence comprising at least one VH of the immunoconjugate or antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising a VL of the immunoconjugate or antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising a VH of the immunoconjugate or antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell. In one embodiment, a method of making a PD-1 -regulated IL-2 immunoconjugate or antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the immunoconjugate or antibody, as provided above, under conditions suitable for expression of the immunoconjugate or antibody, and optionally recovering the immunoconjugate or antibody from the host cell (or host cell culture medium). For recombinant production of a PD-1 -regulated IL-2 immunoconjugate or antibody, nucleic acids encoding the immunoconjugate or antibody, e.g., as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acids may be readily isolated and sequenced using conventional procedures {e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the immunoconjugate or antibody) or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expression of vectors {e.g., expression vectors) include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the immunoconjugate or antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors {e.g., expression vectors), including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, T.U., Nat. Biotech. 22 (2004) 1409- 1414; and Li, H. et a!., Nat. Biotech. 24 (2006) 210-215.

Suitable host cells for the expression of (glycosylated) antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293T cells as described, e.g., in Graham, F.L. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, J.P., Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1 ); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells (as described, e.g., in Mather, J.P. et al., Annals N.Y. Acad. Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub, G. et a!., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

In one aspect, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NSO, Sp20 cell).

I. Assays

In some aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody (e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein may be identified, screened for, or characterized for their physical/chemical properties and/or biological activities by various assays known in the art or disclosed herein.

1 . PD-L1/PD-1 Blocking Reporter Assay

In one aspect, an assay is provided to determine the ability of an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) to block the interaction of PD-1 with its ligand PD-L1 .

In some aspects, a PD-1 binder 7G12 (i.e., AB003058), used to generate several PD-1 regulated IL-2v immunoconjugates described herein, is assessed for blocking the interaction of PD-1 with its ligand PD-L1 , and it is compared in potency to a Roche anti-PD-1 binder.

The PD-1/PD-L1 Blockade Bioassay from Promega (Cat.# J1250, J1255) is used, which consists of a bioluminescent cell-based assay that can be used to measure the potency of antibodies designed to block the PD-1/PD-L1 interaction in a timely fashion.

The assay consists of two genetically engineered cell lines:

PD-1 effector cells: Jurkat T cells expressing a human PD-1 and a luciferase reporter gene driven by an NFAT response element (NFAT-RE); and

PD-L1 antigen-presenting cells (APC)/CHO-K1 cells: CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate T cell receptors (TCRs) in an antigen-independent manner.

When the two cell types are co-cultured, the PD-1/PD-L1 interaction inhibits TCR signaling and nuclear factor of activated T-cells (NFAT)-reporter-mediated luminescence. Addition of either an anti-PD-1 antibody or an anti-PD-L1 antibody that blocks the PD-1 /PD-L1 interaction releases the inhibitory signal and results in TCR activation and NFAT-reporter mediated luminescence.

5 x 10³ PD-L1 -expressing CHO-K1 cells are seeded in 96 well plates overnight, while 5 x 10⁴ PD-1 -expressing Jurkat cells are plated on the day of the experiment. Eight 10-fold dilutions (100 pg/ml -10 pg/ml) of either parental anti-PD-1 blocking antibody (bivalent Roche anti-PD-1 (0376)), an isotype control or 7G12 based PD-1 regulated IL-2v constructs are added to the plate containing the Jurkat reporter cell line, 30 minutes before co-culturing the two cell lines to block the PD-1/PD-L1 pathway. After 6 hours of incubation at 37 °C, the substrate (Bio-Gio Reagent) is added before measuring the samples at the luminometer (TECAN Infinite M1000 Pro). Blockade of PD-1 signaling is measured by the luminescent signal after addition of BIO-GLO™ Luciferase Assay Substrate.

2. Binding assays

In one aspect, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody (e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) as described herein is tested for its antigen binding activity, e.g., by known methods such as ELISA, Western blot, etc.

In some aspects, a clear 384 well plate (Corning # 3700) is coated with 25 pl anti-PGLALA hulgG1 with wildtype Fc (VH and VL are murine, from hybridoma, Roche inhouse) at 1 pg/ml in coating buffer and is incubated overnight at 4 °C. After washing the plate 3 times with 90 pl/well washing buffer on a EL406 BIOTEK® washer, 90 pl blocking buffer is added to the plate and incubated at room temperature for 1 hour. After washing the plate as described above, 25 pl sample (e.g., containing the immunoconjugate) at a concentration of 6 nM in ELISA diluent is added to the plate and followed by another 1 -hour incubation at room temperature. After another washing step, 25 pl biotinylated human IL-2R beta gamma heterodimer, Fc, Avitag (Aero # ILG-H82F3) dilution series, starting at a top concentration of 100 nM, serially diluted in ELISA diluent 1 :3 for 12 points, is added to the plate. After 1 -hour incubation at room temperature, the plate is washed 3 times with 90 pl/well washing buffer on a EL406 BIOTEK® washer and 25 pl Streptavidin-horseradish-peroxidase (POD) (Roche # 11089153001 ) 1 :5000 diluted in ELISA diluent is added to all wells of the plate followed by another 1 -hour incubation time at room temperature and another washing step as described above. 25 pl 1 -Step Ultra TMB substrate solution (Thermo Fisher # 34029) is added, followed by a 5-minute incubation time at room temperature, and measuring optical density (OD) at 370/492 nm using a Tecan Satire 2 multimode reader.

DPBS pH 7.4 (PAN BIOTECH # P04-36500) was used as coating buffer. Superblock pH 7,4 (Thermo Fisher # 37515) is used as blocking buffer. 1 x PBS pH 7.4 (Roche # 11666789001 ) containing 0.5% bovine serum albumin (BSA) and 0.05% TWEEN® 20 is used as diluent for ELISA. 1 x PBS pH 7.4 (Roche #11666789001 ) containing 0.05% TWEEN® 20 is used as washing buffer.

3. IL-2R Signaling Assay

In one aspect, an assay is provided to determine the potency and cis/trans-signaling of an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate (e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti- PD-1 antibody moiety that binds to PD-1 )) .

For this purpose, CD4 T cells from healthy donor PBMCs are sorted with CD4 beads (130- 045-101 , Miltenyi) and activated for 3 days in presence of 1 pg/ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells are harvested and washed several times to remove endogenous cytokines and half of the cells are labeled with Cell Trace Violet (CTV) (5 pM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half are left unlabeled.

Then, the unlabeled cells are incubated with a saturating concentration of a competing anti- PD-1 antibody (in-house molecule, 10 pg/ml) for 30 minutes at RT followed by several washing steps to remove the excess unbound anti-PD-1 antibody. Thereafter, the PD-1 pre-blocked cells (25 pl, 6x10⁶ cells/ml) are co-cultured 1 :1 with the PD-1 ⁺ CTV-labeled cells (25 pl, 6x10⁶ cells/ml) in a V- bottom plate before being treated for 60 minutes at 37 °C with increasing concentrations of treatment immunoconjugates (50 pl, 1 :10 dilution steps). To preserve the phosphorylation state, an equal amount of Phosphoflow Fix Buffer I (100 pl, 557870, BD Bioscience) is added after 60 minutes incubation with the various constructs to allow the unfolding of the PD-1 -regulated IL-2v upon binding to PD-1 . The cells are then incubated for an additional 30 minutes at 37 °C before being permeabilized overnight at -80 °C with Phosphoflow PermBuffer III (558050, BD Bioscience). On the next day, STAT-5 in its phosphorylated form is stained for 30 minutes at 4 °C by using an anti-STAT- 5P antibody (47/Stat5(pY694) clone, 562076, BD Bioscience).

The cells are acquired at the fluorescence-activated cell sorting (FACS) BD-Symphony A5 (BD Bioscience) instrument. The frequency of STAT-5P is determined with FLOWJO™ (V10) and plotted with GraphPad Prism.

The dose-response curves on PD-1 + T cells provide information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 mediated delivery, show the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v the level of leakiness of the Dual Binding Antibody (DBA).

4. Suppression Assay

In one aspect, an assay is provided to assess whether immunoconjugates (e.g., PD-1 - regulated IL-2 immunoconjugates) can reverse the regulatory T cell (Treg) suppression of conventional T cell (T_Conv) effector functions. In some instances, T_COnv and T_reg are isolated and labeled.

In certain aspects, CD4⁺ CD25+ CD127^dim Treg are isolated with the two-step Regulatory T cell Isolation Kit (Miltenyi, #130-094-775). In parallel, the CD4⁺ CD25- T_COnv are isolated by collecting the negative fraction of a CD25 positive selection (Miltenyi, #130-092-983) followed by a CD4⁺ enrichment (Miltenyi, #130-045-101 ). The T_COnv are labeled with carboxyfluoroscein succinimidyl ester (CFSE; eBioscience, #65-0850-84) and the Treg are labeled with Cell Trace Violet (CTV, ThermoFisher Scientific, C34557) to track the proliferation of both populations. T_COnv and T_reg are then cultured together for 5 days, with or without treatment, in presence of CD4- CD25- PBMCs from an unrelated donor to provide an allospecific stimulation.

In certain aspects, on day 5, the accumulation of cytokines in the Golgi complex is enhanced by applying Protein Transport Inhibitors (GOLGIPLUG™ #555029, BD Bioscience; and GOLGISTOP™ #554724, BD Bioscience) for 5 hours prior to the FACS staining. The ability of the proliferated T_COnv to secrete granzyme B (GrzB) in presence and absence of T_reg is measured. T_reg suppression is calculated with the following formula:

. . _ _

% cytokine suppression = 100

Where % cytokine(Tcwv+Treg±immunocwjugate) is the level of cytokine secreted by T_COnv in the presence of Treg ± treatment immunoconjugate and % cytokine<Tconv) is the level of cytokine secreted by T_Conv in the absence of Treg. P is calculated using one-way ANOVA (*p<0.05, **p<0.01 , ***p<0.001 , ****p<0.0001 ).

5. Binding Competition Assay on Activated T Cells

In one aspect, the invention provides an assay to assess whether the blocking and nonblocking DBAs of the immunoconjugates (e.g., PD-1 -regulated-IL-2 immunoconjugates) competes with the blocking and non-blocking anti-PD-1 targeting arm, a binding competition was performed on PD-1 expressing (PD-1 +) activated CD4 T cells.

For this purpose, CD4 T cells are sorted from healthy donor PBMCs with CD4 beads (130- 045-101 , Miltenyi) and activated for 3 days in presence of 1 pg/ml plate bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells are harvested and washed before exposing them to increasing concentrations of treatment antibodies (50 pl, 1 :10 dilution steps) for 30 minutes at 4 °C. The treated cells, after two washing steps, are stained for additional 30 minutes at 4 °C with saturating concentrations of blocking and non-blocking dual antibodies (/.e., DBA moiety) directly labeled with ALEXA FLUOR®-647 and Life/Dead Fixable viability Dye eFluor 780 before fixation. Cells are then acquired on a FACS BD-Symphony A5 (BD Bioscience) instrument, FACS analyses are performed using FLOWJO (V10), and the data are plotted with GraphPad Prism.

6. Competitive Binding Assay

In one aspect, the invention provides an assay to assess whether the DBA moieties and/or anti-PD-1 antibody moieties have different epitopes on the surface of PD-1 or if they compete for the same or overlapping binding epitopes.

Surface plasmon resonance (SPR) experiments are performed on a BIACORE® T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KCI, 1 .37 M NaCI and 0.5% Surfactant P20 (TWEEN® 20), Cytiva, Freiburg, Germany). A CAP chip (provided in the Biotin CAPture Kit, series S, Cytiva, 28920234) pre-coated with an ssDNA oligo sequence is used in this experiment, normalized and hydrodynamically addressed, according to the manufacturer’s instructions.

Initially, the Biotin CAPture Reagent containing the complementary oligo sequence to which streptavidin was immobilized is sent overflow cells 1 and 2. Hybridization of complementary ssDNA- SA to pre-immobilized ssDNA oligo on the CAP-chip is performed for 300 sec at a flow rate of 2 pL/min. The biotinylated human PD-1 (internal ID P1 AF8774) is then injected for 60 seconds with a flow rate of 10 pL/min at a concentration of 25 nM.

To measure the competitive binding to PD-1 , first the PD-1 -binding molecule (e.g., containing DBA moieties or anti-PD-1 antibody moieties) is injected for 180 sec with a flow rate of 10 pL/min at a concentration of 10 pg/mL. Second, directly after the first injection, a different PD-1 -binding molecule (e.g., containing DBA moieties or anti-PD-1 antibody moieties) with 10 mg/ml is injected on both flow cells for 180 sec at a flow speed of 10 pL/min. Dissociation time is set to 120 sec followed by a stabilization period of 60 sec.

An increasing signal during the second injection phase indicates a different epitope region of the first PD-1 -binding molecule bound on PD-1 compared to the second, because this construct does not block the binding epitope of the second injected PD-1 -binding molecule to PD-1 . No additional binding signal indicates the blocking of the second PD-1 -binding molecule to PD-1 because the epitope region is bound by the first PD-1 -binding molecule. Blocking indicates that the two PD-1 - binding molecules bound competitively to the same epitope.

The Biotin CAPture Reagent, as well as the bound analyte, is then removed from the surface after each analysis cycle using a mixture of three parts of Regeneration Stock 1 (8 M guanidine-HCI) with 1 part of Regeneration Stock 2 (1 M NaOH) provided in the Biotin CAPture Kit. The regeneration solution mixture is injected for 120 seconds at a flow rate of 5 pL/min following a stabilization period of 90 sec.

7. Competitive Binding Assay

In one aspect, the invention provides an assay to detect IL-2-signaling triggered by the IL-2 moiety in an PD-1 -regulated IL-2 immunoconjugate.

Material

Cell clones: PD-1 -expressing cell clones are internally generated using the HEK-Blue IL-2 reporter cell line from InvivoGen. Three clones are created expressing high (clone 42), medium (clone 26) and low (clone 4) PD-1 levels. Assay media and reagents: DMEM 4.5 g/L, 10 % FBS, 2 mM L-Glutamine, Trypsin (Pan Biotech P10-023100), QUANTI-BLUE™ Solution (InvivoGen Cat# rep- qbs2). Protocol

Molecules were diluted in cell media to reach a maximum assay concentration of 20 nM when testing HEK-Blue IL-2 WT reporter cells (non-PD-1 expressing) and a maximum assay concentration of 2 nM when testing HEK-Blue IL-2 reporter PD-1 clones. 8 serial dilutions at 1 :8 dilution were prepared and 25 pl/well was pipetted in 384-well flat bottom transparent plates. Afterwards, the four cell lines (HEK-Blue IL-2 WT, HEK-Blue IL-2 PD-1 clones 42, 26 and 4) were trypsinized for 3 minutes at 37 °C and resuspended in assay media at a concentration of 4x10⁵ cells/mL. Next, 25 pl/well of cell suspension was added to the previously prepared plates containing the molecules reaching a cell density of 1 x10⁴ cells/well. Plates were placed in the incubator for 24 hours. QUANTI-BLUE™ reagent was prepared following the manufacturer’s instructions. Briefly, QUANTI-BLUE™ reagent was thawed at room temperature, incubated for 2 minutes at 37 °C in a water bath to dissolve possible crystals, and resuspended in 98 mL sterile water plus 1 mL QUANTI-BLUE™ buffer. The diluted QUANTI- BLUE™ solution was immediately used or stored at -20 °C. 45 pl/well of QUANTI-BLUE™ solution was pipetted in 384-white transparent flat bottom plates. Plates containing the cell treatments were taken from the incubator and left at room temperature for 10 min. Afterwards, 5 pl of cell supernatant was carefully acquired and pipetted onto the plates containing the QUANTI-BLUE™reagent using the Viaflow Assist Plus (Integra). Plates were incubated for 20-30 minutes in an orbital shaker (300 rpm at RT) and absorbance was measured on an absorbance reader (Tecan) using the following settings: read type: Endpoint, wavelength: 620 nm.

8. Colorimetric Assay

HEK-Blue™ IL-2 reporter cells are specifically designed to detect human IL-2/IL-15 by the activation of the JAK-STAT pathway by stable transfection of HEK293 cells with the human CD25 (IL- 2Ra), CD122 (IL-2R0), and CD132 (IL-2Ry) genes, along with the human JAK3 and STAT5 genes to obtain a fully active IL-2 signaling pathway. In addition, a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene is also introduced to the reporter cells. Upon IL-2 stimulation, HEK-Blue™ IL-2 cells trigger the JAK/STAT5 activation and the subsequent secretion of SEAP, which can then be readily monitored using QUANTI-Blue™ Solution.

Cells are cultured and prepared as per manufacturer’s instructions, and exposed to dose increasing concentrations of IL-2 immunoconjugates for 24 hours at 37°C. The supernatant of the cultured cells is then harvested and placed in a flat bottom plate together with QUANTI-Blue Solution for 6 hours before measuring SEAP levels using a spectrophotometer at 620-655 nm.

Dose-response curves depicting the absorbance measured at the spectrophotometer indicate the activity of the immunoconjugates in absence of PD-1 expression due to high avidity binding of the IL-2v for the high density of IL-2R per single cell.

J. Methods and Compositions for Diagnostics and Detection

In certain aspects, an anti-PD-1 antibody provided herein is useful for detecting the presence of PD-1 in a biological sample. The term “detecting” as used herein encompasses quantitative or qualitative detection. In certain aspects, a biological sample comprises a cell or tissue, such as tumor.

In one aspect, an anti-PD-1 antibody for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of PD-1 in a biological sample is provided. In certain aspects, the method comprises contacting the biological sample with an anti-PD-

1 antibody as described herein under conditions permissive for binding of the anti-PD-1 antibody to PD-1 , and detecting whether a complex is formed between the anti-PD-1 antibody and PD-1 . Such method may be an in vitro or in vivo method.

In certain aspects, a labeled anti-PD-1 antibody is provided. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron- dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵l, ³H, and ¹³¹1, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, p-galactosidase, glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

K. Pharmaceutical Compositions

In a further aspect, provided are pharmaceutical compositions comprising a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-

2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein, e.g., for use in any of the below therapeutic methods. In one aspect, a pharmaceutical composition comprises a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the immunoconjugates or antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

Pharmaceutical compositions (formulations) of an antibody or a PD-1 -regulated IL-2 immunoconjugate as described herein can be prepared by combining the immunoconjugate or antibody with pharmaceutically acceptable carriers or excipients known to the skilled person. See, for example Flemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1^st Ed., Woodhead Publishing (2015), §4 and Falconer R.J., Biotechnology Advances (2019), 37, 107412. Exemplary pharmaceutical compositions of an antibody or a PD-1 - regulated IL-2 immunoconjugate as described herein are lyophilized, aqueous, frozen, and the like.

Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as histidine, phosphate, citrate, acetate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes {e.g., Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).

The pharmaceutical composition herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide an additional therapeutic agent useful for treatment of the same disease. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

The pharmaceutical compositions to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

L. Therapeutic Methods and Routes of Administration

In some aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) provided herein may be used in therapeutic methods.

In one aspect, a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti- PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) for use as a medicament is provided. In further aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) for use in treating cancer is provided. In certain aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) for use in a method of treatment is provided. In certain aspects, the present disclosure provides a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) for use in a method of treating a subject {e.g., a human subject) in need thereof comprising administering to the subject an effective amount of the immunoconjugate or antibody.

In a further aspect, the present disclosure provides for the use of a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL- 2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) in the manufacture or preparation of a medicament. In one aspect, the medicament is for treatment of cancer. In a further aspect, the medicament is for use in a method of treating cancer comprising administering to the subject {e.g., a human subject) in need thereof an effective amount of the medicament. In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, e.g., as described below.

In a further aspect, the present disclosure provides a method for treating cancer. In one aspect, the method comprises administering to a subject {e.g., a human subject) in need thereof an effective amount of a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti- PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) in order to treat the cancer. In one such aspect, the method further comprises administering to the subject an effective amount of at least one additional therapeutic agent, as described below.

Exemplary cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, bladder cancer (e.g., urothelial carcinoma (UC), including metastatic UC (mUC); muscle-invasive bladder cancer (MIBC), and non-muscle-invasive bladder cancer (NMIBC)); kidney or renal cancer (e.g., renal cell carcinoma (RCC)); lung cancer, including small-cell lung cancer, nonsmall cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; cancer of the urinary tract; breast cancer (e.g., HER2+ breast cancer and triple-negative breast cancer (TNBC), which are estrogen receptors (ER-), progesterone receptors (PR-), and HER2 (HER2-) negative); prostate cancer, such as castration-resistant prostate cancer (CRPC); cancer of the peritoneum; hepatocellular cancer; gastric or stomach cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)); glioblastoma; cervical cancer; ovarian cancer; liver cancer (e.g., hepatocellular carcinoma (HCC)); hepatoma; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine carcinoma; salivary gland carcinoma; prostate cancer; vulval cancer; thyroid cancer; hepatic carcinoma; anal carcinoma; penile carcinoma; melanoma, including superficial spreading melanoma, lentigo malignant melanoma, acral lentiginous melanomas, and nodular melanomas; multiple myeloma and B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myologenous leukemia (AML); hairy cell leukemia; chronic myeloblastic leukemia (CML); post-transplant lymphoproliferative disorder (PTLD); and myelodysplastic syndromes (MDS), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), Meigs’ syndrome, brain cancer, head and neck cancer, and associated metastases.

In certain aspects, the cancer is bladder cancer, blood cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, and skin cancer.

In certain aspects, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, head and neck cancer, liver cancer, lung cancer, or skin cancer.

In certain aspects, the cancer is a solid tumor cancer.

In certain aspects, the cancer is a PD-1 -positive cancer.

In a further aspect, the present disclosure provides pharmaceutical compositions comprising any of the immunoconjugates or the antibodies described herein, e.g., for use in any of the above therapeutic methods. In one aspect, a pharmaceutical composition comprises any of the immunoconjugates or the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition comprises any of the immunoconjugates or the antibodies provided herein and at least one additional therapeutic agent, e.g., as described below.

In some aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) as described herein can be administered alone or used in a combination therapy, e.g., useful in treating cancer. For instance, the combination therapy includes administering a immunoconjugate or an antibody as described herein and administering at least one additional therapeutic agent {e.g., one, two, three, four, five, or six additional therapeutic agents).

The at least one additional therapeutic agent encompasses any agent that can be administered for treatment. In certain aspects, the additional therapeutic agent is an additional anticancer agent. Exemplary anti-cancer agents include, but are not limited to, a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA intercalator, an alkylating agent, a hormonal therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, antiangiogenic agent, an immunomodulatory agent, an inhibitor of cell adhesion, a cytotoxic or cytostatic agent, an activator of cell apoptosis, an agent that increases the sensitivity of cells to apoptotic inducers, a cytokine, an anti-cancer vaccine or oncolytic virus, a toll-like receptor (TLR) agent, a bispecific antibody, a cellular therapy, and immune cell engager. In certain aspects, the additional therapeutic agent is an immunomodulatory anti-cancer agent, e.g., a checkpoint inhibitor (CPI) such as an anti-CTLA4 antibody {e.g., ipilimumab), a PD-L1 binding antagonist, or a PD-1 binding antagonist.

A PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti- PD-1 antibody moieties) as described herein (and any additional therapeutic agent) can be administered by any suitable means, including parenterally, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, by inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g. liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference). The administration may be systemic or local. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

In some aspects, an immunoconjugate (e.g., a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 )) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) as described herein can be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject species being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The immunoconjugate or the antibody need not be, but is optionally formulated with, one or more agents currently used to treat the disorder in question. The effective amount of such other agents depends on the amount of immunoconjugate or antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clin ically determined to be appropriate. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs.

In some examples, a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti-PD-1 antibody moieties) may be suitably administered to the subject at one time or over a series of treatments. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

M. Articles of Manufacture

In another aspect, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture may include a PD-1 -regulated IL-2 immunoconjugate {e.g., including at least one binding domain that binds to PD-1 conjugated to an IL-2 polypeptide; e.g., including a dual binding antibody (DBA) moiety that binds to PD-1 and an IL-2 polypeptide in a mutually exclusive manner and an anti-PD-1 antibody moiety that binds to PD-1 ) and/or an antibody {e.g., including two DBA moieties or including two anti- PD-1 antibody moieties) disclosed herein. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an immunoconjugate or antibody as disclosed herein. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises an antibody as disclosed herein; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture in this aspect as described herein may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

Example 1. Generation of PD-1 -regulated IL-2 Immunoconjugates

1.1. Construction of expression plasmids for immunoconjugates

For the expression of immunoconjugates as reported herein, a transcription unit comprising the following functional elements was used:

• the immediate early enhancer and promoter from the human cytomegalovirus (P- CMV) including intron A,

• a human heavy chain immunoglobulin 5’-untranslated region (5’UTR),

• a murine immunoglobulin heavy chain signal sequence,

• a nucleic acid encoding the respective fusion polypeptide, and

• the bovine growth hormone polyadenylation sequence (BGH pA).

Beside the expression unit/cassette including the desired gene, the basic/standard mammalian expression plasmid contains:

• an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and

• a beta-lactamase gene which confers ampicillin resistance in E. coli.

1.2. Expression of immunoconjugates

Transient expression of immunoconjugates was performed in suspension-adapted Expi293 (EXPI293F™ cells; Thermofisher Scientific) with EXPIFECTAMINE™ 293 Transfection Kit (EXPIFECTAMINE™ 293 Reagent, EXPIFECTAMINE™ 293 Transfection Enhancers 1 and 2).

Cells were passaged, by dilution, at least four times (volume 30 ml) after thawing in a 125-ml shake flask (incubated/shaked at 37 °C, 7% CO2, 85% humidity, 135 rpm). The cells were expanded to 3x10⁵ cells/ml in 250 ml volume. Three days later, cells were split and newly seeded with a density of 1 .5x10⁶ to 7x10⁵ cells/ml in a 250 ml volume in a 1 -liter shake flask. T ransfection was performed 24 hours later at a cell density around 1 .4 - 3.0x10⁶ cells/ml.

Before transfection, 250 pg plasmid-DNA was diluted in a final volume of 12.5 ml with preheating (water bath; 37 °C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for up to 5 min. For transfections with EXPIFECTAMINE™, 675 pl of the EXPIFECTAMINE™ 293 reagents were added to 12.5 ml OptiMEM-solution and incubated in a separate tube for 5 minutes. Next, the two solutions were united, mixed gently, and incubated at room temperature for 15-20 minutes, and the complete mixture was added to a 1 -L shake flask with 250 ml Expi293 cell culture.

The incubation was performed by shaking the flask at 37 °C, 7% CO2, 85% humidity, 135 rpm for 6 to 7 days.

The supernatant was harvested by filtration through diatomaceous earth Sartoclear Dynamics Lab Filter Aid 10g, Product no. SDLKG-10.0 - 2 (Sartorius Stedim Biotech), subsequently filtered through a 0.22-pm bottle top filter and stored in a freezer (-20 °C).

1.3. Purification of immunoconjugates

The immunoconjugate-containing culture supernatants were filtered and purified by two chromatographic steps. The immunoconjugates were captured by affinity chromatography using HITRAP® MABSELECT™ SURE™ affinity antibody columns (GE Healthcare) equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCI, 2.7 mM KCI), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and the immunoconjugates were recovered with 100 mM acetate buffer, pH 2.8, and immediately after elution neutralized to pH 6.0 with 1 M Tris-base, pH 9.0. Subsequently, size exclusion chromatography on a SUPERDEX 200™ column (GE Healthcare) was used as polishing step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCI, pH 6.0. Finally, the immunoconjugate-containing solutions were concentrated with an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80 °C.

1.4. Mass spectrometric analysis of the immunoconjugates

PNGase F was obtained from Roche Diagnostics GmbH (14.3 U/pl; solution in sodium phosphate, EDTA and glycerol).

Enzymatic deglycosylation of with PNGase F

100 pg of immunoconjugates was diluted to a final concentration of 0.5 mg/ml with 100 mM sodium phosphate buffer, pH 7.1 , and deglycosylated with 1 pl PNGase F at 37°C for 16 hours.

ESI-QTOF mass spectrometry The digested samples were then analyzed by LC-MS. Liquid chromatography was performed on a Waters Acquity ultra-performance liquid chromatography (UPLC) (Waters) with a reversed-phase C18 column (Agilent PLRP-S column, 2.1 x 150 mm, 8 pm, 1000A (Agilent, Cat. -Nr.: PL1912-3802)). The aqueous mobile phase (mobile phase A) contained 0.1% (v/v) formic acid (FA) in HPLC grade water. The organic mobile phase (mobile phase B) contained 0.1 % FA in acetonitrile. The gradient that was utilized in this experiment is plotted in Table 9 below:

Table 9. HPLC program for purification of immunoconjugates.

Further chromatographic settings:

Flow rate: 0.6 mL/min

Temperature of the column oven: 75 °C

Injection volume: 8 pl

The UPLC was coupled to an electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS) instrument (maXis II UHR-QTOF MS system (Bruker Daltonik)). Calibration was performed with sodium iodide. For the digested immunoconjugate, data acquisition was done at 800-4000 m/z (isCID: 85 eV). The raw mass spectra were evaluated and transformed into individual relative molar masses. For visualization of the results proprietary software was used to generate deconvoluted mass spectra. Yields are shown below in Table 10.

Table 10. Purification yield of transiently expressed immunoconjugates. opt: affinity matured; di: de-immunized; crossed: crossover modification; dicrossed = de-immunized and crossover modification; BO: bottle-opener

As compared to P1 AI7440, P1 AJ1837 has Asp at the C-terminus of the DBA light chain, which has no impact on the binding properties. Thus, these molecules can be considered equivalent in terms of activity. The same applies to P1AJ1838 and P1AI7441.

Example 2. 7G12 Anti-PD-1 Antibody (/.e., AB003058) Blocks the PD-1-PD-L1 Pathways in PD-1 Jurkat Reporter Assay and Is 10-fold Less Potent than a Bivalent Anti-PD-1 Blocking Antibody (/.e., 0376)

The PD-1 binder 7G12 (/.e., AB003058), used to generate several PD-1 regulated IL-2v immunoconjugates described herein, was assessed for blocking the interaction of PD-1 with its ligand PD-L1 , and it was compared in potency to a Roche anti-PD-1 binder.

The PD-1/PD-L1 Blockade Bioassay from Promega (Cat.# J1250, J1255) was used, which consists of a bioluminescent cell-based assay that can be used to measure the potency of antibodies designed to block the PD-1/PD-L1 interaction in a timely fashion.

The assay consists of two genetically engineered cell lines:

PD-1 effector cells: Jurkat T cells expressing a human PD-1 and a luciferase reporter gene driven by an NFAT response element (NFAT-RE); and

PD-L1 antigen-presenting cells (APC)/CHO-K1 cells: CHO-K1 cells expressing human PD-L1 and an engineered cell surface protein designed to activate cognate T cell receptors (TCRs) in an antigen-independent manner. When the two cell types were co-cultured, the PD-1/PD-L1 interaction inhibited TCR signaling and nuclear factor of activated T-cells (NFAT)-reporter-mediated luminescence. Addition of either an anti-PD-1 antibody or an anti-PD-L1 antibody that blocked the PD-1/PD-L1 interaction released the inhibitory signal and resulted in TCR activation and NFAT-reporter mediated luminescence.

5 x 10³ PD-L1 -expressing CHO-K1 cells were seeded in 96 well plates overnight, while 5 x 10⁴ PD-1 -expressing Jurkat cells were plated on the day of the experiment. Eight 10-fold dilutions (100 pg/ml -10 pg/ml) of either parental anti-PD-1 blocking antibody (bivalent Roche anti-PD-1 (0376)), an isotype control or 7G12 based PD-1 regulated IL-2v constructs were added to the plate containing the Jurkat reporter cell line, 30 minutes before co-culturing the two cell lines to block the PD-1/PD-L1 pathway. After 6 hours of incubation at 37 °C, the substrate (Bio-Gio Reagent) was added before measuring the samples at the luminometer (TECAN Infinite M1000 Pro). Blockade of PD-1 signaling was measured by the luminescent signal after addition of BIO-GLO™ Luciferase Assay Substrate.

All 7G12-based constructs showed activity in the PD-1/PD-L1 blockade bioassay, indicating that 7G12 blocked the interaction of PD-1 with PD-L1 , leading to the dose response increase of the relative luminescent unit. In addition, 7G12 was shown to have 10-fold higher EC50 than Roche anti- PD-1 , indicative of a 10-fold lower potency (FIG. 1 and Table 11). As expected, the isotype control did not elicit any effect on the luminescence of the reporter cell line.

Table 11. EC50 of PD-1/PD-L1 blockage.

Example 3. IL-2R Signaling (STAT5-P) on Activated PD-1⁺ and PD-1' CD4 T Cells upon Treatment with Increasing Doses of PD-1-Regulated IL-2v AF5842

The potency and the cis/trans-signaling of a PD-1 -regulated IL-2v (PD-1 -regulated IL-2 immunoconjugate) were measured as IL-2R signaling by treating activated PD-1 expressing (PD-1+) and PD-1 negative (PD-1 (anti-PD-1 pre-treated) CD4 T cells with increasing concentrations of immunoconjugates. The purpose was to determine the dependency of the PD-1 -regulated IL-2v on the PD-1 expression of the T cells in order to deliver IL-2R signaling.

For this, CD4 T cells from healthy donor PBMCs were sorted with CD4 beads (130-045-101 , Miltenyi) and activated for 3 days in presence of 1 pg/ml plate-bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and half of the cells were labeled with Cell Trace Violet (CTV) (5 pM, 5 minutes at room temperature (RT); C34557, Thermo Scientific) and the other half were left unlabeled. Then, the unlabeled cells were incubated with a saturating concentration of a competing anti- PD-1 antibody (in-house molecule, 10 ig/ml) for 30 minutes at RT followed by several washing steps to remove the excess unbound anti-PD-1 antibody. Thereafter, the PD-1 pre-blocked unlabeled cells (25 pl, 6*10⁶ cells/ml) were co-cultured 1 :1 with the PD-1 ⁺ CTV-labeled cells (25 pl, 6x10⁶ cells/ml) in a V-bottom plate before being treated for 60 minutes at 37 °C with increasing concentrations of treatment immunoconjugates (50 pl, 1 :10 dilution steps). To preserve the phosphorylation state, an equal amount of Phosphoflow Fix Buffer I (100 pl, 557870, BD Bioscience) was added after 60 minutes incubation with the various constructs to allow the unfolding of the PD-1 -regulated IL-2v upon binding to PD-1 . The cells were then incubated for an additional 30 minutes at 37 °C for fixation before being permeabilized overnight at -80 °C with Phosphoflow PermBuffer III (558050, BD Bioscience). On the next day, STAT-5 in its phosphorylated form was stained for 30 minutes at 4 °C by using an anti-STAT-5P antibody (47/Stat5(pY694) clone, 562076, BD Bioscience).

The cells were acquired at the flow cytometer (FACS) BD-Symphony A5 (BD Bioscience) instrument. The frequency of STAT-5P was determined with FLOWJO (V10) and plotted with GraphPad Prism.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 mediated delivery, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v the level of leakiness of the Dual Binding Antibody (DBA). In this particular assay, AF5842 was shown to be 8-fold less potent than PD-1 -IL-2v (always-on, e.g., always-on P1 AE4422-14542; see PCT Publication No. WO 2018/184964, which is incorporated herein by reference in its entirety), however it has a roughly 100-fold higher activity on PD-1+ than on PD-1 ■ T cells, as opposed to the 80-fold of PD-1 -IL-2v (FIG. 2 and Table 12). As expected, FAP-IL-2v (see PCT Publication No. WO 2012/146628, which is incorporated herein by reference in its entirety) was 50-fold less potent than PD-1 -IL-2v, lacked cis-activity, and therefore was equally active on T cells regardless of their PD-1 expression.

Table 12. IL-2R signaling activity upon treatment with increasing doses of immunoconjugate.

Example 4. IL-2R Signaling (STAT5-P) on Activated PD-1⁺ and PD-1- CD4 T Cells upon Treatment with Increasing Doses of Various PD-1 -Regulated IL-2v Containing One or Two IL-2v with DBAs with or without One or More Anti-PD-1 Targeting Arms The potency and the cis/trans-signaling of several PD-1 -regulated IL-2v were measured as IL- 213 signaling by treating activated PD-1 ⁺ and PD-1 - (anti-PD-1 pre-treated) CD4 T cells with increasing concentration of immunoconjugates. The purpose was to determine the dependency of the PD-1 - regulated IL-2v on the PD-1 expression of the T cells in order to deliver IL-2R signaling. CD4 T cells were processed as described in Example 3 for FIG. 2, and treated for 12 or 60 minutes at 37 °C with increasing concentrations of treatment immunoconjugates.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent PD-1 -mediated delivery, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v, the level of leakiness of the DBA. In this particular assay, AF5842 was shown to be 22-fold less potent than PD-1 -IL-2v after 60 minutes of incubation, however it had more than 200-fold higher activity on PD-1 ⁺ than on PD-1 - T cells. The PD-1 -regulated-IL-2v molecules were shown to have a more than 300-fold reduction in potency when compared with PD-1 - IL-2v after 60 minutes and had reduced cis-activity (as EC50 fold increase: EC50 PD-1 blocked/ EC50 PD-1 ⁺) when compared with AF5842 and PD-1 -IL-2v (FIG. 3A and FIG. 3B and Table 13).

FAP-IL-2v was roughly 80-fold less potent than PD-1 -IL-2v and lacked cis-activity, and therefore it was equally active on T cells regardless of their PD-1 expression.

As an additional control, the frequency of STAT-5P+ T cells in the PD-1 + and PD-T exposed to incremental doses of PD-1 -IL-2v for 12 minutes was also included. At 60 minutes of incubation, the potency of PD-1 -IL-2v increased roughly 10-fold, resulting in more than two-fold higher cis-activity on PD-1 ⁺ T cells than after 12 minutes of incubation.

Table 13. IL-2R signaling activity upon treatment with different immunoconjugates.

Example 5. IL-2R signaling (STAT5-P) on Activated PD-1+ and PD-1- CD4 T Cells upon

T eatment with Increasing Doses of Various PD-1 -Regulated IL-2v Containing One IL-2v with Blocking or Non-Blocking DBAs and One or More Anti-PD-1 Targeting Arms with Blocking or Non-Blocking Activity

The potency and the cis/trans-signaling of various PD-1 -regulated IL-2v were measured as the ability to elicit IL-2R signaling in activated PD-1 ⁺ and PD-1 - (anti-PD-1 pre-treated) CD4 T cells by exposing them to increasing concentration of immunoconjugates. The purpose was to determine the conditional and selective activity of the PD-1 -regulated IL-2v relative to the PD-1 expression on the T cells in order to deliver IL-2R signaling in cis. For this purpose, CD4 T cells were processed as described in Example 3, and treated for 12 or 60 minutes at 37 °C with increasing concentrations of treatment immunoconjugates.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent PD-1 docking, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v, the level of leakiness of the DBA. In this particular assay, the PD-1 -regulated- IL-2v molecules were shown to have between 5- and 100-fold reduction in potency when compared with PD-1 -IL-2v after 60 minutes and some of them were shown to have an improved cis-activity (as EC50 fold increase: EC50 PD-1 blocked/ EC50 PD-1 + or as AUC delta) when compared with AF5842 and PD-1 -IL-2v (FIG. 4A and FIG. 4B and Table 14, and FIG. 5A and FIG. 5B and Table 15).

Table 14. IL-2R signaling activity upon treatment with different immunoconjugates. bl.: blocked

Table 15. IL-2R signaling activity upon treatment with different immunoconjugates.

bl.: blocked

AF5842 was roughly 20-fold less potent than PD-1 -IL-2v after 60 minutes of incubation; however, it had more than 100-fold higher activity on PD-1 ⁺ than on PD-1 - T cells. FAP-IL-2v was less potent than PD-1 -I L-2v, lacked cis-activity, and therefore it was equally active on T cells regardless of their PD-1 expression.

As an additional control, the frequency of STAT-5P⁴ T cells in the PD-1 + and PD-1 - exposed to incremental doses of PD-1 -IL-2v for 12 minutes was also included. At 60 minutes of incubation the potency of PD-1 -IL-2v increased roughly 5-fold, resulting in roughly 2-fold higher cis-activity on PD-1 ⁺ T cells than after 12 minutes (FIG. 4).

Example 6. IL-2R signaling (STAT5-P) on Activated PD-1⁺ and PD-1- CD4 T Cells Upon T eatment with Increasing Doses of Various PD-1 -Regulated IL-2v Containing One IL-2v with Blocking or Non-Blocking DBAs and One Anti-PD-1 Targeting Arm with Blocking or Non- Blocking Activity

The potency and the cis/trans-signaling of various PD-1 -regulated IL-2v were measured as the ability to elicit IL-2R signaling in activated PD-1 ⁺ and PD-1 - (anti-PD-1 pre-treated) CD4 T cells by exposing them to increasing concentration of immunoconjugates. The purpose was to determine the conditional and selective activity of the PD-1 -regulated IL-2v relative to the PD-1 expression on the T cells in order to deliver an IL-2R signaling in cis. CD4 T cells were processed as described in Example 3 and treated for 12 or 60 minutes at 37°C with increasing concentrations of treatment immunoconjugates.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 docking, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v the level of leakiness of the DBA. In this particular assay, the PD-1 -regulated- IL-2v molecules were shown to have between 6- and 600-fold reduction in potency when compared with PD-1 -IL-2v after 60 minutes and some of them were shown to have an improved cis-activity (as EC50 fold increase: EC50 PD-1 blocked/ EC50 PD-1 + or as AUC delta) when compared with AF5842 and PD-1 -IL-2V (FIG. 6 and Table 16).

AF5842 was roughly 100-fold less potent than PD-1 -IL-2v after 60 minutes of incubation, however, it had more than 400-fold higher activity on PD-1 + than on PD-1 - T cells. As expected, FAP- IL-2v was less potent than PD-1 -IL-2v, lacked cis-activity, and therefore it was equally active on T cells regardless of their PD-1 expression.

As an additional control, the frequency of STAT-5P⁴ T cells in the PD-1 + and PD-1 - exposed to incremental doses of PD-1 -IL-2v for 12 minutes was also included. At 60 minutes of incubation, the potency of PD-1 -IL-2v increased roughly 10-fold resulting in roughly two-fold higher cis-activity on PD- 1 + T cells than after 12 minutes (FIG. 6 and Table 16).

Table 16. IL-2R signaling activity upon treatment with different immunoconjugates. bl.: blocked

Example 7. Functional Activity of Various PD-1-regulated IL-2v (PD-1-reg-IL-2v) on Cytotoxic Effector Functions and Proliferation of Allo-Specific PD-1 ⁺ CD4 T Cells

To assess the functional activity of PD-1 -reg-IL-2v on the effector functions of T cells and compare it to PD-1 -IL-2v, FAP-IL-2v, and AF5842, CD4 T cells were co-cultured for 5 days with a 13- cell lymphoblastoid tumor cell line (ARH77) to generate allo-reactive T cells. ARH77 expresses intermediate levels of PD-L1 and high levels of MHCII and induces PD-1 expression on the surface of the allo-specific CD4 T cells (FIG. 7A). This assay therefore allows for the functional assessment of PD-1 blockade and PD-1 -mediated delivery of regulated IL-2v.

CD4 isolation and CTV labelling was conducted as described above. The sorted CD4⁺ T cells were co-cultured with irradiated ARH-77 (human B lymphoblast cell line) in an E:T ratio of 5:1 (100,000 T Cells: 20,000 ARH-77) in the presence or absence of increasing doses of PD-1 -based or FAP-based IL-2 constructs. The cells were co-cultured in a 96-round bottom plate for 5 days at 37 °C, 5% CO2. After 5 days the accumulation of cytokines in the Golgi complex was enhanced by applying Protein Transport Inhibitors (GOLGIPLUG™, 555029, BD Bioscience; and GOLGISTOP™, 554724, BD Bioscience) for 5 hours prior to the FACS staining. The cells were first stained for CD4 and for live/dead. After fixation/permeabilization overnight (554714, BD Bioscience), the cells were stained intracellularly for Granzyme B (GrzB). The cells were acquired at the FACS BD-Symphony A5 (BD Bioscience) instrument and analyzed with FLOWJO® and GRAPHPAD PRISM®. By gating on the living and proliferating CD4⁺ T cells (CTV^low), the frequency and mean fluorescence intensity of granzyme B secretion was compared between the conditions.

The dose-response curves indicated that the PD-1 -reg-IL-2v were functionally active and similarly potent in eliciting cytotoxic T cell effector functions. They showed at various degrees a lower activity on T cell effector functions than PD-1 -IL-2v and a higher one than FAP-IL-2v (FIG. 7 and Table 17).

Table 17. Cytotoxic T cell effector function of different immunoconjugates as determined by granzyme B secretion from CD4 T cells.

Example 8. Rescue of T_COnv Effector Function from T_reg Suppression upon PD-1-reg-IL-2v Treatment

To assess whether PD-1 -reg-IL-2v constructs can reverse the regulatory T cell (T_reg) suppression of conventional T cell (T_COnv) effector functions, a suppressive-function assay was established. For this purpose, T_COnv and Treg were isolated and labeled. Briefly, CD4⁺ CD25⁺ CD127^dim Tre were isolated with the two-step Regulatory T cell Isolation Kit (Miltenyi, #130-094-775). In parallel, the CD4⁺ CD25- T_COnv were isolated by collecting the negative fraction of a CD25 positive selection (Miltenyi, #130-092-983) followed by a CD4⁺ enrichment (Miltenyi, #130-045-101 ). The T_COnv were labeled with CFSE (eBioscience, #65-0850-84) and the Treg were labeled with CTV (ThermoFisher Scientific, C34557) to track the proliferation of both populations. T_Conv and Treg were then cultured together for 5 days, with or without treatment, in presence of CD4- CD25- PBMCs from an unrelated donor to provide an allospecific stimulation.

On day 5, the accumulation of cytokines in the Golgi complex was enhanced by applying Protein Transport Inhibitors (GOLGIPLUG™ #555029, BD Bioscience; and GOLGISTOP™ #554724, BD Bioscience) for 5 hours prior to the FACS staining and acquisition on a FACS BD-Symphony A5 (BD Bioscience) instrument. Data were analyzed with FLOWJO (V10) and data were plotted using with GraphPad Prism. The ability of the proliferated T_COnv to secrete granzyme B (GrzB; FIG. 8A and FIG. 8B) in presence and absence of T_reg was measured. The Treg suppression was calculated with the following formula:

. . _ _

% cytokine suppression = 100 where % cytokine(Tconv+Treg±immunoconjugate) is the level of cytokine secreted by T_COnv in the presence of Treg ± treatment immunoconjugate and % cytokine<Tconv) is the level of cytokine secreted by T conv in the absence of Treg.

In untreated samples, more than 95% of granzyme B secretion by T_COnv was suppressed by Treg . Treatment with untargeted IL-2v (FAP-IL-2v) did not significantly rescue T_COnv secretion of granzyme B from Treg suppression. On the contrary, the treatment with PD-1 -IL-2v, used as positive control due to its ability to overcome Treg suppression, resulted in a pronounced reduction of Treg suppression to 48%, while the AF5842 molecule had a modest reduction to 71% (FIG. 8B).

A portion of the PD-1 -reg-IL-2v was shown to significantly overcome Treg suppression when compared to the untreated group (FIG. 8A and FIG. 8B and Table 18).

Table 18. Percentage of suppression of T_COnv Granzyme B secretion by T_reg.

Example 9. IL-2R Signaling (STAT5-P) on Activated PD-1⁺ and PD-1' CD8 T Cells Upon Treatment with Increasing Doses of Various PD-1 -Regulated IL-2v Constructs Containing One Additional Amino Acid at C-Terminus of the DBA Light Chain The potency and the cis/trans-signaling of various PD-1 -regulated IL-2v constructs with the introduction of one additional amino acid at the C-terminus of the DBA light chain were measured and compared with the parental molecules for the ability to elicit IL-2R signaling in activated PD-1+ and PD-1- (anti-PD-1 pre-treated) CD8 T cells. The purpose was to determine whether the addition of an amino acid at the C-terminus of the sequence of the DBA light chain might affect the conditional and selective activity of the PD-1 -regulated IL-2v constructs relative to the PD-1 expression on the T cells in order to deliver IL-2R signaling in cis.

For this purpose, CD8 T cells were sorted from healthy donor PBMCs with CD8 beads (130- 045-201 , Miltenyi), and processed as described for CD4 in Example 3 above for FIG. 2, and treated for 12 or 60 minutes at 37 °C with increasing concentrations of treatment antibodies.

In this particular assay, the PD-1 -regulated-IL-2v molecules containing an additional amino acid at the C-terminus were shown to have comparable or slightly improved potency and cis-activity as the parental molecules (FIG. 9A - FIG. 9C and Table 19).

Table 19. IL-2R signaling activity upon treatment with different immunoconjugates. bl = blocking

As compared to P1 AI7440, P1 AJ1837 has Asp at the C-terminus of the DBA light chain, which has no impact on the binding properties. Thus, these molecules can be considered equivalent in terms of activity. The same applies to P1 AJ1838 and P1 AI7441 . Example 10. Conditional Activity of PD-1-reg-IL-2v Constructs on PD-1⁺ T Cells over NK Cells to Assess the Leakiness of Dual Blocking Antibodies

To assess whether PD-1 -reg-IL-2v constructs were conditionally active on PD-1 + T cells and inactive on PD-T cells like NK cells, a dose-response competition assay was performed on activated T cells co-cultured at 1 :5 ratio with freshly isolated PBMCs.

For this purpose, CD4 and CD8 T cells were sorted from healthy donor PBMCs with CD4 beads (130-045-101 , Miltenyi) and with CD8 beads (130-045-201 , Miltenyi), activated for 3 days in presence of 1 pg/ml plate bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells were harvested and washed several times to remove endogenous cytokines and the cells were labeled with CTV (5 pM, 5 minutes at room temperature; C34557, Thermo Scientific).

On the same day of the labelling, 10⁶ freshly isolated PBMCs were stained with CD16 plus CD56, and after several washing steps, were co-cultured for 60 minutes at 37 °C with 2 x 10⁵ labeled activated CD4 and CD8 T cells in the presence of increasing concentrations of treatment antibodies (50 pl, 1 :10 dilution steps). The cells were then incubated for additional 30 minutes at 37 °C with an equal amount of PHOSPHOFLOW® Fix Buffer I (100 pl, 557870, BD Bioscience) to preserve the phosphorylation state, before being stained with surface linage markers followed by overnight permeabilization at -80 °C with PHOSPHOFLOW® PermBuffer III (558050, BD Bioscience). On the next day STAT-5 in its phosphorylated form was stained for 30 minutes at 4 °C by using an anti-STAT-5P antibody (47/Stat5(pY694) clone, 562076, BD Bioscience).

The cells were acquired on a FACS BD-Symphony A5 (BD Bioscience) instrument. The frequency of STAT-5P was determined with FLOWJO (V10) and plotted with GraphPad Prism.

PD-1 -IL-2v was roughly 10-fold more potent in eliciting IL-2R signaling on PD-1 expressing CD4 (FIG. 10A) and CD8 T cells (FIG. 10B) than on fresh NK cells (FIG. 10C). However, the reference molecule AF5842, although 10- to 15-fold less potent on PD-1 + CD4 and CD8 T cells than PD-1 -IL-2v, was virtually inactive on NK cells (FIG. 10A - FIG. 10D).

Results are summarized below in Table 20.

Table 20. Conditional Activity of Immunoconjugates on Activated CD4/CD8 cells and NK cells. Notably, three PD-1 -reg-IL-2v constructs were shown to have only roughly 2- to 3-fold reduced potency in signaling through the IL-2R on PD-1 + T cells than PD-1 -IL-2v. However, these molecules were inactive on NK cells, similar to the reference molecule AF5842 (FIG. 10A - FIG. 10C). Only the P1 AJ2535 construct (which contains 2 mutations in the VH and VL instead of one in the VL) displayed activity on NK cells indicating the leakiness of the non-blocking DBA after amino acids substitution during the de-immunization.

Example 11. Binding Competition of Blocking and Non-Blocking Dual Binding Antibodies with Blocking and Non-Blocking Anti-PD-1 Targeting Antibodies

To assess whether the blocking and non-blocking DBAs of the PD-1 -reg-IL-2v constructs competed with the blocking and non-blocking anti-PD-1 targeting arm, a binding competition was performed on PD-1 expressing (PD-1 +) activated CD4 T cells.

For this purpose, CD4 T cells were sorted from healthy donor PBMCs with CD4 beads (130- 045-101 , Miltenyi) and activated for 3 days in presence of 1 pg/ml plate bound anti-CD3 (overnight pre-coated, clone OKT3, #317315, BioLegend) and 1 pg/ml of soluble anti-CD28 (clone CD28.2, #302923, BioLegend) antibodies to induce PD-1 expression. Three days later, the cells were harvested and washed before exposing them to increasing concentrations of treatment antibodies (50 pl, 1 :10 dilution steps) for 30 minutes at 4 °C. The treated cells, after two washing steps, were stained for additional 30 minutes at 4 °C with saturating concentrations of blocking and non-blocking dual antibodies directly labeled with ALEXA FLUOR®-647 and Life/Dead Fixable viability Dye eFluor 780 before fixation. Cells were then acquired on a FACS BD-Symphony A5 (BD Bioscience) instrument, FACS analyses were performed using FLOWJO (V10), and the data were plotted with GraphPad Prism.

The blocking DBA AB002345 competed for binding on PD-1 + activated CD4 T cells with the blocking anti-PD-1 antibody clone 0376 which occupied 80% of the PD-1 receptors, while blocking DBA AB002345 did not compete with the non-blocking DBA AB003637 nor with the non-blocking anti- PD-1 antibody clone 1040. As a positive control, the same blocking DBA AB002345 was used to prevent the binding of the directly labeled blocking DBA AB002345 achieving similar occupancy as clone 0376 (FIG. 11 A).

Conversely, the non-blocking DBA AB003637 competed for binding on PD-1 + activated CD4 T cells with the non-blocking anti-PD-1 antibody clone 1040, with the blocking DBA AB002345, and with the non-blocking DBA AB003637 used as positive control, while it did not compete with the blocking anti-PD-1 antibody clone 0376 (FIG. 11B).

Example 12. Ligand Inhibition/Competitive Assays for PD-1/IL-2v Immunoconjugates

Ligand Inhibition Assay

The aim of the ligand inhibition assay was to determine if the binding of the antibodies (DBA and anti-PD-1 ) to PD-1 inhibits the binding between PD-L1 and PD-1. SPR experiments were performed on a BIACORE® T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KCI, 1 .37 M NaCI and 0.5% Surfactant P20 (TWEEN® 20), Cytiva, Freiburg, Germany).

A CAP chip (provided in the Biotin CAPture Kit, series S, Cytiva, 28920234) pre-coated with an ssDNA oligonucleotide (oligo) sequence was used in this experiment, normalized and hydrodynamically addressed, according to the manufacturer’s instructions.

Initially, the Biotin CAPture Reagent containing the complementary oligo sequence to which streptavidin was immobilized was sent over flow cells 1 and 2. Hybridization of complementary ssDNA-SA to pre-immobilized ssDNA oligo on the CAP-chip is performed for 360 sec at a flow rate of 2 pL/min. The biotinylated human PD-1 (internal ID P1AF8774) was then injected for 180 seconds with a flow rate of 5 pL/min at a concentration of 100 nM.

To measure the ligand inhibition, first the PD-1 molecule was injected for 300 sec with a flow rate of 5 pL/min at a concentration of 10 pg/mL. Second, directly after the first injection, a constant amount of PD-L1 (internal ID P1 AD8568, 10OOnM), which was mixed with 10 mg/ml of the same PD- 1 -binding molecule , was injected on both flow cells for 150 sec at a flow speed of 5 pL/min. No dissociation time was necessary.

An increasing signal during the second injection phase was observed for P1 AG3741 and P1 AI7514, which indicates for these binders an epitope region on PD-1 which did not interfere with the interaction between PD-L1 and PD-1 . No additional binding signal was observed for P1 AI3784 and P1 AH4157, which indicates the blocking of PD-L1 binding to PD-1 .

The Biotin CAPture Reagent, as well as the bound analyte, were then removed from the surface after each analysis cycle using a mixture of three parts of Regeneration Stock 1 (8 M guanidine-HCI) with 1 part of Regeneration Stock 2 (1 M NaOH) provided in the Biotin CAPture Kit. The regeneration solution mixture was injected for 120 seconds at a flow rate of 5 pL/min following a stabilization period of 90 sec. A summary of the ligand inhibition ability of various immunoconjugates is shown below in Table 21 .

Table 21. Summary of Ligand Inhibition Assay on Immunoconjugates. Competitive Binding Assay

The aim of the competitive binding experiment was to determine if the binders have different epitopes on the surface of PD-1 or if they compete for the same or overlapping binding epitopes.

Surface plasmon resonance (SPR) experiments were performed on a BIACORE® T200 at 25 °C with PBS-P+ as running and sample dilution buffer (0.2 M phosphate buffer with 27 mM KCI, 1 .37 M NaCI and 0.5% Surfactant P20 (TWEEN® 20), Cytiva, Freiburg, Germany).

A CAP chip (provided in the Biotin CAPture Kit, series S, Cytiva, 28920234) pre-coated with an ssDNA oligo sequence was used in this experiment, normalized and hydrodynamically addressed, according to the manufacturer’s instructions.

Initially, the Biotin CAPture Reagent containing the complementary oligo sequence to which streptavidin was immobilized was sent over flow cell 1 and 2. Hybridization of complementary ssDNA-SA to pre-immobilized ssDNA oligo on the CAP-chip was performed for 300 sec at a flow rate of 2 pL/min. The biotinylated human PD-1 (internal ID P1 AF8774) was then injected for 60 seconds with a flow rate of 10 pL/min at a concentration of 25 nM.

To measure the competitive binding to PD-1 , first the PD-1 -binding molecule (e.g., DBA moiety or anti-PD-1 antibody moiety) was injected for 180 sec with a flow rate of 10 pL/min at a concentration of 10 pg/mL. Second, directly after the first injection, a different PD-1 -binding molecule (e.g., DBA moiety or anti-PD-1 antibody moiety) with 10 mg/ml was injected on both flow cells for 180 sec at a flow speed of 10 pL/min. Dissociation time was set to 120 sec followed by a stabilization period of 60 sec.

An increasing signal during the second injection phase indicated a different epitope region of the first PD-1 -binding molecule bound on PD-1 compared to the second, because this construct did not block the binding epitope of the second injected PD-1 -binding molecule to PD-1 . No additional binding signal indicated the blocking of the second PD-1 -binding molecule to PD-1 because the epitope region was bound by the first PD-1 -binding molecule. Blocking indicated that the two PD-1 - binding molecules bound competitively to the same epitope.

The Biotin CAPture Reagent, as well as the bound analyte, was then removed from the surface after each analysis cycle using a mixture of three parts of Regeneration Stock 1 (8 M guanidine-HCI) with 1 part of Regeneration Stock 2 (1 M NaOH) provided in the Biotin CAPture Kit. The regeneration solution mixture is injected for 120 seconds at a flow rate of 5 pL/min following a stabilization period of 90 seconds. The results of the competitive binding assay are shown in Table 22 below.

Table 22. Summary of Competitive Binding Assay on Immunoconjugates.

+ Competition observed; - No competition observed

Example 13. Enzyme-Linked Immunosorbent Assay (ELISA)-Based PD-1-reg-IL-2v: IL-2Rpy Binding Assay

The regulation ELISA described in this Example assessed the switch behavior of the PD-1 - regulated IL-2 constructs dependent on the PD-1 concentration.

Buffer:

DPBS pH 7.4 (PAN BIOTECH # P04-36500) was used as coating buffer. Superblock pH 7,4 (Thermo Fisher # 37515) was used as blocking buffer. 1 x PBS pH 7.4 (Roche # 11666789001 ) containing 0.5% bovine serum albumin (BSA) and 0.05% TWEEN® 20 was used as diluent for ELISA. 1x PBS pH 7.4 (Roche #11666789001 ) containing 0.05% TWEEN® 20 was used as washing buffer.

Method:

A clear 384 well plate (Corning # 3700) was coated with 25 pl anti-PGLALA hulgG1 with wildtype Fc (VH and VL are murine, from hybridoma, Roche inhouse) at 1 pg/ml in coating buffer and incubated overnight at 4 °C. After washing plate 3 times with 90 pl/well washing buffer on a EL406 BIOTEK® washer, 90 pl blocking buffer was added to the plate and incubated at room temperature for 1 hour. After washing the plate as described above 25 pl sample at a concentration of 6 nM in ELISA diluent was added to the plate and followed by another 1 -hour incubation at room temperature. After another washing step, 25 pl biotinylated human IL-2R beta gamma heterodimer, Fc, Avitag (Aero # ILG-H82F3) dilution series, starting at a top concentration of 100 nM, serially diluted in ELISA diluent 1 :3 for 12 points, was added to the plate. After 1 -hour incubation at room temperature, the plate was washed 3 times with 90 pl/well washing buffer on a EL406 BIOTEK® washer and 25 pl Streptavidin- horseradish-peroxidase (POD) (Roche # 11089153001 ) 1 :5000 diluted in ELISA diluent was added to all wells of the plate followed by another 1 -hour incubation time at room temperature and another washing step as described above. 25 pl 1 -Step Ultra TMB substrate solution (Thermo Fisher # 34029) was added, followed by a 5-minute incubation time at room temperature, and measuring optical density (OD) at 370/492 nm using a Tecan Satire 2 multimode reader.

Results of the ELISA assay of various immunoconjugates are shown in FIG. 12A and FIG. 12B. The “always-off” control did not possess any IL-2 module within the molecule and therefore did not bind IL-2R. The “always-on” control was a fusion of a PD-1 binder with I L-2v, and therefore showed PD-1 -independent IL-2R binding. The PD-1 -regulated IL-2 constructs exhibited PD-1 - dependent binding to IL-2R.

Example 14. HEK-Blue IL-2 Reporter Assay

The HEK-Blue IL-2 reporter cell line is engineered to detect IL-2-signali ng which in this assay is triggered by the IL-2 moiety in the PD-1 -regulated IL-2 constructs. HEK-Blue IL-2 reporter cells were used to generate 3 cell lines expressing different levels of PD-1 . These 3 cell lines plus the WT cell line were used in this assay to detect the difference between the off state and the on state of the PD-1 -regulated IL-2 constructs in dependence on PD-1 expression level.

Material

Cell clones: PD-1 -expressing cell clones were internally generated using the HEK-Blue IL-2 reporter cell line from InvivoGen. 3 clones were created expressing high (clone 42), medium (clone 26) and low (clone 4) PD-1 levels. Assay media and reagents: DMEM 4.5 g/L, 10 % FBS, 2 mM L- Glutamine, Trypsin (Pan Biotech P10-023100), QUANTI-BLUE™ Solution (InvivoGen Cat# rep-qbs2).

Protocol

Molecules were diluted in cell media to reach a maximum assay concentration of 20 nM when testing HEK-Blue IL-2 WT reporter cells (non-PD-1 expressing) and a maximum assay concentration of 2 nM when testing HEK-Blue IL-2 reporter PD-1 clones. 8 serial dilutions at 1 :8 dilution were prepared and 25 pl/well was pipetted in 384-well flat bottom transparent plates. Afterwards, the four cell lines (HEK-Blue IL-2 WT, HEK-Blue IL-2 PD-1 clones 42, 26 and 4) were trypsinized for 3 minutes at 37 °C and resuspended in assay media at a concentration of 4x10⁵ cells/mL. Next, 25 pl/well of cell suspension was added to the previously prepared plates containing the molecules reaching a cell density of 1 x10⁴ cells/well. Plates were placed in the incubator for 24 hours. QUANTI-BLUE™ reagent was prepared following the manufacturer’s instructions. Briefly, QUANTI-BLUE™ reagent was thawed at room temperature, incubated for 2 minutes at 37 °C in a water bath to dissolve possible crystals, and resuspended in 98 mL sterile water plus 1 mL QUANTI-BLUE™ buffer. The diluted QUANTI- BLUE™ solution was immediately used or stored at -20 °C. 45 pl/well of QUANTI-BLUE™ solution was pipetted in 384-white transparent flat bottom plates. Plates containing the cell treatments were taken from the incubator and left at room temperature for 10 min. Afterwards, 5 pl of cell supernatant was carefully acquired and pipetted onto the plates containing the QUANTI-BLUE™ reagent using the Viaflow Assist Plus (Integra). Plates were incubated for 20-30 minutes in an orbital shaker (300 rpm at RT) and absorbance was measured on an absorbance reader (Tecan) using the following settings: read type: Endpoint, wavelength: 620 nm.

Results are shown in FIG. 13A - FIG. 13M. The “always-on” control (FIG. 13A) was a regular fusion of IL-2 to a PD-1 binder without any switch function and therefore its IL-2 activity did not depend on PD-1 . The always-on control showed high IL-2 signaling activation even in the WT cells and increased signaling in PD-1 expressing cells due to the retention of the molecule on the cell surface by PD-1 binding. All the switch molecules showed low activity on WT cells as the molecules were switched off by shielding of the IL-2 fusion by the DBA in the absence of PD-1 . In the case of the PD-1 expressing cells, the binding of the PD-1 -regulated IL-2 molecules to PD-1 led to the exposure of the IL-2 fusion, which then became free to trigger IL-2 signaling activation in the reporter cells. The PD-1 -regulated IL-2 immunoconjugates showed a much stronger IL-2 activity in the clones than in the WT cell line. This difference was much smaller in the always-on control, where the increased activity in the clones was only due to higher retention of molecule and not due to a switch mechanism. The fact that IL-2 activation of the PD-1 -regulated IL-2 immunoconjugates depended on PD-1 levels was very well reflected in the case of P1 AI7455 and P1 AI7441 , which showed a correlation between IL-2 activation and PD-1 levels, the high-PD-1 expressing cells being the most responsive to these PD-1 -regulated IL-2 immunoconjugates.

Example 15. In Vivo Efficacy of PD-1-reg-IL-2v as Single Agents in a Syngeneic Model of Mouse Tumor Cell Line.

PD-1 -reg-IL-2v immunoconjugate bispecific antibodies were tested as single agents for their anti-tumoral efficacy in a syngeneic model.

Panc02-Fluc Pancreatic Subcutaneous Syngeneic Model

The murine surrogate PD-1 -reg-IL-2v immunoconjugates were tested in the mouse pancreatic Panc02-Fluc cell line subcutaneously injected into Black 6-huPD tg mice.

Panc02-Fluc cell line was produced in house by calcium transfection and sub-cloning techniques. Panc02-Fluc was cultured in RPMI medium containing 10% fetal calf serum (FCS) (Sigma), 500 pg/ml hygromicin and 1 % of GLUTAMAX®. The cells were cultured at 37 °C in a water- saturated atmosphere at 5 % CO2. Passage 15 was used for transplantation. Cell viability was 96.4%. 2x10⁵ cells per animal were injected subcutaneously in 100 pl of RPMI cell culture medium (Gibco) into the flank of mice using a 1 ml tuberculin syringe (BD Biosciences).

Female Black 6-huPD-1 tg mice, aged 8-10 weeks at the start of the experiment (Charles Rivers, Lyon, France), were maintained under specific-pathogen-free condition with daily cycles of 12 hour light / 12 hour darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 184/2020). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.

Mice were injected subcutaneously on study day 0 with 2x10⁵ of Panc02-Fluc cells, randomized and weighed. Thirteen days after the tumor cell injection (tumor volume > 120 mm³), mice in the different groups were treated as follow (see Table 23):

1 - i.v. with mu1 +1 DBAnon-cross_AB002345_FV020519_IL-2v_4G2SG2 (P1 AI8597) twice a week for one week

2.- i.v. with mu1 +1 DBAnon-cross_AB003637_FV020519_IL-2v_4G2SG2 (P1 AI8600) twice a week for one week

3- i.v. with mu1 +1 DBAcrossCH1 Ck_AB002345_FV014541_IL-2v_4G2SG2 (P1 AI8565) twice a week for one week

4- i.v. with mu1 +1 DBAcrossCFH Ck_AB002345_FV020519_IL-2v_4G2SG2 (P1 AI8564) twice a week for one week

5- i.v. with mu1 +1 scFv_AB002345_AB003058_IL-2v_4G2SG2_4G2SG2 (P1 AI8589) twice a week for one week 6- i.v. with huPD-1 -mulgG1 -DAPD-DD/KK with mulL-2v (muPD-IL-2v) (P1AE2791 ) twice a week for one week

7- i.v. with Pembrolizumab twice a week for one week

8- i.v. with Histidine Buffer

Table 23. Treatment conditions for groups 1-7.

All mice injected intravenously (i.v.) received 200 pl of the appropriate solution (e.g., containing the appropriate dose of immunoconjugate/antibody). The mice in the Vehicle group (i.e., group 8) were injected with 200 pl of histidine buffer and the treatment groups with the PD-1 -reg-IL-2v constructs at 10 mg/kg or muPD-1 -IL-2v 0.5 mg/kg or pembrolizumab at 10 mg/kg (Table 23). To obtain the proper amount of immunoconjugate per 200 pl, stock solutions were diluted with Histidine Buffer. Tumor growth was followed by caliper measurement and data were transferred to Graph Pad for visualization and statistical analysis. Tumor growth data are shown in FIG. 14.

3 days after the last administration, sentinel mice were sacrificed for each group (4 mice/group) and the lung weight was evaluated as shown in FIG. 15. For this measurement, the lungs were excised and placed in a balance and the weights were measured and recorded for comparison between groups using Graph Pad Prism.

Additionally, immuno-pharmacodynamic (Immuno-PD) analysis by flow cytometry was performed on tumors and blood of each group (4 mice/group) as shown in FIG. 16A and FIG. 16B, which were harvested from the same sentinel mice 3 days after the last therapy administration. The tumors were processed into small pieces and were digested with collagenase D and DNAse I for 30 minutes at 37 °C in order to derive a single cell suspension. Tumor single cell suspensions and blood were stained with directly labeled antibodies (CD45-APC-Cy7, TCRb-BUV737, CD8-BV510, CD4- BUV395, R&D, Switzerland). Samples were acquired using a BD Fortessa flow cytometer. Following analysis with FLOWJO version 10.1 , results were visualized with Graph Pad Prism.

As shown in FIG. 14, the PD-1 -reg-IL-2v constructs mediated superior efficacy in terms of tumor growth inhibition compared to the vehicle and pembrolizumab groups. It also shows that for most PD-1 -reg-IL-2v constructs, except for P1 AI8600, the anti-tumor efficacy is similar to muPD-1 -IL- 2v molecule.

As shown in FIG. 15, the lung weights of the muPD-1 -IL-2v treated mice were significantly increased 3 days after the last therapy treatment. However, the lung weights of the PD-1 -reg-IL-2v treated mice, except for the P1 AI8565 construct, were comparable to vehicle and pembrolizumab- treated mice, indicating lack of peripheral activity for the PD-1 -reg-IL-2v molecules.

As shown in FIG. 16A and FIG. 16B, muPD-1 -IL-2v and PD-1 -reg-IL-2v impacted treatment on CD8-positive (CD8⁺) T cell expansion in tumor (FIG. 16A) and blood (FIG. 16B). Both PD-1 -reg- IL-2v constructs and muPD-1 -IL-2v increased the number of CD8⁺ T cells in the tumor compared to the vehicle and pembrolizumab groups (FIG. 16A). However, in the blood only muPD-1 -IL-2v showed a high significant increase in CD8 T cells (FIG. 16B), indicating lack or minimal peripheral activity for the PD-1 -reg-IL-2v molecules, except for the P1 AI8565 construct, which also showed a smaller increase in blood CD8⁺ T cells.

Example 16. IL-2R signaling (STAT5-P) on Activated PD-1⁺ and PD-1- CD4 T Cells Upon T eatment with Increasing Doses of Various PD-1 -Regulated IL-2v Containing One IL-2v with Blocking or Non-Blocking DBAs and One Anti-PD-1 Targeting Arm with Blocking or Non- Blocking Activity

The potency and the cis/trans-signaling of various PD-1 -regulated IL-2v were measured as the ability to elicit IL-2R signaling in activated PD-1 ⁺ and PD-1 - (anti-PD-1 pre-treated) CD4 T cells by exposing them to increasing concentration of immunoconjugates. The purpose was to determine the conditional and selective activity of the PD-1 -regulated IL-2v relative to the PD-1 expression on the T cells in order to deliver an IL-2R signaling in cis. CD4 T cells were processed as described in Example 3 and treated for 60 minutes at 37°C with increasing concentrations of treatment immunoconjugates.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R on PD-1 + T cells. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 docking, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and in the case of the PD-1 -regulated IL-2v the level of leakiness of the DBA. The ratio between the EC50 on PD-1 T cells versus the PD-1 + T cells or the delta AUC between the PD-1 + and the PD-1 ■ T cells represent the cis-activity window of the tested molecules and therefore the ability to bind to PD-1 and signal through the IL-2R on the same cell. In this particular assay, the PD-1 - regulated-IL-2v was shown to have 32-fold reduction in potency on PD-1 • T cells when compared with PD-IL-2v after 60 minutes of incubation and to have an improved cis-activity (as EC50 fold increase: EC50 PD-1 blocked/ EC50 PD-1 + or as AUC delta) when compared PD-1 -IL-2v (FIG. 17A and Table 24).

As expected, FAP-IL-2v was less potent than PD-1 -IL-2v, lacked cis-activity, and therefore it was equally active on T cells regardless of their PD-1 expression (FIG. 17A and Table 24).

The 1 +1 format of the PD-1 -reg-IL-2v with one DBA and one PD-1 only binder, in which IL-2v is attached to the DBA, was compared to the asymmetric format of Compound C with two DBAs, in which IL-2v is attached to one DBA (2B07 IL-2 mut in Example 18 and Figure 20 of WO 2021/231773). Compound C was roughly 10-fold less potent than PD-1 -IL-2v after 60 minutes of incubation, however, it had more than 200-fold higher activity on PD-1 + than on PD-T T cells. However, although PD-1 -reg-IL-2v only exhibited two-fold less potent than PD-1 -IL-2v on PD-1 + T cells, PD-1 -reg-IL-2v showed a 1 100-fold higher activity on PD-1 ⁺ in comparison to PD-1 - T cells and therefore had a 5- and 15-folds higher cis-activity window than Compound C and PD-1 -IL-2v respectively (FIG. 17A and Table 24). These results illustrate the superiority of the 1 +1 format of the PD-1 -reg-IL-2v with one DBA and one PD-1 only binder (e.g., anti-PD-1 antibody moiety) compared to the asymmetric format with two DBAs (e.g., wherein one DBA is conjugated to an IL-2 polypeptide and one DBA is not) in increasing the cis-activity and overall potency on PD-1 + cells. This better cis window increases the therapeutic index of the molecule. The asymmetric format with two DBAs exhibited a reduced cis-window and overall lower potency as higher EC50 value on PD-1 positive T cells than the 1 +1 format. This is explained partly by the choice of a PD-1 targeting binder (e.g., anti- PD-1 antibody moiety) with higher affinity. Also, systemic free IL-2 could play a role in the reduction of activity in the asymmetric format by binding the DBA in the targeting arm, which interferes with the PD-1 binding of the DBA in the targeting arm.

Table 24. IL-2R signaling activity upon treatment with different immunoconjugates.

Importantly, when comparing PD-1 -reg-IL-2v with PD-1 -reg-IL-2vQ126T, which contains an additional mutation in the IL-2v to further decrease the binding affinity for IL-2Rb and therefore increase the conditional activity of the IL-2v on the PD-1 expression, it was observed that PD-1 -reg- IL-2vQ126T was equally potent to PD-1 -reg-IL-2v on PD-1 + T cells. However, PD-1 -reg-IL-2vQ126T did not show any activity on PD-1 - T cells indicating that the additional mutation in the IL-2v (e.g., Q126T) coupled with the masking of the DBA were able to completely prevent the leaky binding to and signaling of the IL-2R, e.g., in the absence of PD-1 expression. (FIG. 17B and Table 25).

Table 25. IL-2R signaling activity upon treatment with different immunoconjugates. bl.: blocked

Example 17. Conditional Activity of PD-1-reg-IL-2v Constructs on PD-1⁺ T Cells over NK Cells to Assess the Leakiness of Dual Blocking Antibodies

To assess whether PD-1 -reg-IL-2v constructs were conditionally active on PD-1 + T cells and inactive on PD-1 - cells like NK cells, which however express 10-fold higher IL-2Rb than T cells, a dose-response competition assay was performed on activated T cells co-cultured at 1 :5 ratio with freshly isolated PBMCs. CD4 T cells, CD8 T cells, and NK cells were processed as described in Example 10 and treated for 60 minutes at 37°C with increasing concentrations of treatment immunoconjugates.

PD-1 -IL-2v was more than 100-fold more potent in eliciting IL-2R signaling on PD-1 expressing CD4 (FIG. 18A) and CD8 T cells (FIG. 18B) than on fresh NK cells (FIG. 18C). PD-1 -reg- IL-2v and PD-1 -reg-IL-2vQ126T showed only 2-8 fold reduced activity on PD-1 + CD8 and CD4 T cells than PD-1 -IL-2v. However PD-1 -reg-IL-2v had some degree of activity on NK cells, indicating leakiness of the DBA, although this leakiness was strongly reduced when compared to PD-1 -IL-2v and FAP-IL-2v. Importantly PD-1 -reg-IL-2vQ126T was completely inactive on NK cells supporting the selective conditional activity of the immunoconjugate, e.g., that activity of the I L-2v, is tightly regulated by PD-1 expression (FIG. 18A - FIG. 18C).

Results are summarized below in Table 26.

Table 26. Conditional Activity of Immunoconjugates on Activated CD4/CD8 cells and NK cells. Data shown as EC50. Example 18 Colorimetric Change in the Substrate Upon Treatment with Increasing Doses of PD-1 Regulated IL-2v on HEK-Blue™ IL-2 Reporter Cell Line.

To assess the leakiness of the mask of the DBA under unphysiological conditions a HEK- Blue™ IL-2 reporter cell line expressing roughly 30-fold more IL-2R beta than activated PD-1 + T cells was used.

HEK-Blue™ IL-2 reporter cells have been specifically designed to detect human IL-2/IL-15 by the activation of the JAK-STAT pathway by stable transfection of HEK293 cells with the human CD25 (IL-2Ra), CD122 (IL-2R0), and CD132 (IL-2Ry) genes, along with the human JAK3 and STAT5 genes to obtain a fully active IL-2 signaling pathway. In addition, a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene was also introduced. Upon IL-2 stimulation, HEK-Blue™ IL-2 cells triggered the JAK/STAT5 activation and the subsequent secretion of SEAP, which could be readily monitored using QUANTI-Blue™ Solution.

Cells were cultured and prepared as per manufacturer’s instructions, and exposed to dose increasing concentrations of IL-2 immunoconjugates for 24 hours at 37°C. The supernatant of the cultured cells was then harvested and placed in a flat bottom plate together with QUANTI-Blue™ Solution for 6 hours before measuring SEAP levels using a spectrophotometer at 620-655 nm.

The dose-response curves depicting the absorbance measured at the spectrophotometer indicate the activity of the immunoconjugates in absence of PD-1 expression due to high avidity binding of the IL-2v for the high density of IL-2R per single cell. This assay was therefore useful to detect and enhance even weak leakiness of the DBA mask (FIG. 19 and Table 27).

PD-1 -IL-2v and FAP-IL-2v induced a similar dose-response activation of the SEAP element followed by PD-1 -reg-IL-2v with more than 300-fold reduction in potency when compared to PD-1 -IL- 2v. Importantly, PD-1 -reg-IL-2vQ126T did not trigger the activation of the SEAP element, further supporting the conclusion that the DBA mask and the additional mutation of the IL-2v (i.e., Q126T) resulted in the conditional interaction of IL-2v with the IL-2R exclusively in the presence of PD-1 receptors, thereby reducing leakiness of the immunoconjugate (FIG. 19 and Table 27).

Table 27. SEAP activity downstream IL-2R signaling of HEK-Blue™ IL-2 reporter cell line upon treatment with different immunoconjugates.

Example 19. IL-2R signaling (STAT5-P) on Activated PD-1⁺ and PD-1- CD4 T Cells Upon Treatment with Increasing Doses of PD-1-Regulated IL-2vQ126T with 10- and 20-mer Linkers

The potency and the cis/trans-signaling of PD-1 -regulated-IL-2vQ126T with different linker lengths were measured as the ability to elicit IL-2R signaling in activated PD-1 ⁺ and PD-1 - (anti-PD-1 pre-treated) CD4 T cells by exposing them to increasing concentration of immunoconjugates. The purpose was to determine whether the conditional and selective activity of the PD-1 -regulated-IL-2v relative to the PD-1 expression on the T cells was affected by the linker’s length. For this CD4 T cells from healthy donor PBMCs were processed, activated and acquired as described in Example 3.

The dose-response curves on PD-1 + T cells provided information on the potency of the assessed molecules in signaling through the IL-2R on PD-1 + T cells. In addition, the dose-response curves on T cells pre-treated with a competing anti-PD-1 antibody, to prevent the PD-1 docking, showed the potency of the molecules in providing IL-2R signaling independently from PD-1 expression, and therefore the level of leakiness of the DBA. The delta AUC between the PD-1 + and the PD-1 ■ T cells represent the cis-activity window of the tested molecules and therefore the ability to bind to PD-1 and signal through the IL-2R on the same cell.

The PD-1 -regulated-IL-2vQ126T 10-mer and 20-mer linkers showed to have comparable potency on PD-1 ⁺ T cells, no activity on PD-1 - T cells and to have an improved cis-activity window as AUC delta after 60 minutes of incubation (FIG. 20 and Table 28). EC50 data reported as EC50 fold increase. Therefore, the length of the linker did not affect the potency of the PD-1 -regulated-IL- 2vQ126T on PD-1 + T cells as well as its inactivity, or PD-1 dependent conditional activity, on PD-T T cells.

Table 28. IL-2R signaling activity upon treatment with different immunoconjugates. EC50 corresponds to EC50.

Bl.: blocked

EMBODIMENTS

1 . An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide;

(ii) a linker; and

(iii) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus, and -C denotes a polypeptide C-terminus, and wherein the first binding domain is configured such that: (i) when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor; and

(ii) when the DBA is bound to PD-1 , the DBA moiety is substantially blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to an IL-2 receptor; and (b) a second binding domain comprising an anti-PD-1 antibody moiety comprising a VH and a VL.

2. The immunoconjugate of embodiment 1 , wherein the anti-PD-1 antibody moiety does not substantially bind to an IL-2 polypeptide.

3. The immunoconjugate of embodiment 1 or 2, wherein the DBA moiety comprises a Fab molecule.

4. The immunoconjugate of embodiment 3, wherein the DBA moiety comprises a Fab heavy chain comprising the VH of the DBA moiety and a heavy chain constant domain 1 (CH1 ) and a Fab light chain comprising the VL of the DBA moiety and a light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

5. The immunoconjugate of embodiment 3, wherein the DBA moiety is a conventional Fab molecule.

6. The immunoconjugate of any one of embodiments 1 -5, wherein the anti-PD-1 antibody moiety comprises a Fab molecule.

7. The immunoconjugate of embodiment 6, wherein the anti-PD-1 antibody moiety comprises a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

8. The immunoconjugate of any one of embodiments 1 -5, wherein:

(a) the DBA moiety is a conventional Fab molecule; and

(b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

9. The immunoconjugate of any one of embodiments 1 -5, wherein:

(a) the DBA moiety is a conventional Fab molecule; and

(b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other.

10. The immunoconjugate of embodiment 6, wherein the anti-PD-1 antibody moiety is a conventional Fab molecule. 11 . The immunoconjugate of any one of embodiments 1 -5, wherein:

(a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and

(b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

12. The immunoconjugate of any one of embodiments 1 -5, wherein:

(a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and

(b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

13. The immunoconjugate of any one of embodiments 1 -5, wherein the anti-PD-1 antibody moiety is a single-chain variable fragment (scFv).

14. The immunoconjugate of any one of embodiments 1 -13, further comprising an Fc domain comprising a first subunit and a second subunit.

15. The immunoconjugate of any one of embodiments 1 -14, wherein (a) the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus; and/or (b) the anti-PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

16. The immunoconjugate of embodiment 14 or 15, wherein the Fc domain is an IgG Fc domain.

17. The immunoconjugate of embodiment 16, wherein the IgG Fc domain is an IgGi Fc domain.

18. The immunoconjugate of any one of embodiments 14-17, wherein the Fc domain is a human IgG Fc domain.

19. The immunoconjugate of any one of embodiments 14-18, wherein the first subunit comprises one or more CH domains selected from a first CH2 (CH2i) domain and/or a first CH3 (CH3i) domain; and the second subunit comprises one or more CH domains selected from a second CH2 (CH22) domain and/or a second CH3 (CH32) domain.

20. The immunoconjugate of embodiment 19, wherein at least one of the one or more CH domains is paired with another CH domain.

21 . The immunoconjugate of embodiment 20, wherein the CH3i and CH32 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH3i domain is positionable in the cavity or protuberance, respectively, in the CH32 domain.

22. The immunoconjugate of embodiment 21 , wherein the CH3i and CH32 domains meet at an interface between the protuberance and cavity. 23. The immunoconjugate of any one of embodiments 20-22, wherein the CH2i and CH22 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH2i domain is positionable in the cavity or protuberance, respectively, in the CH22 domain.

24. The immunoconjugate of embodiment 23, wherein the CH2i and CH22 domains meet at an interface between the protuberance and cavity.

25. The immunoconjugate of any one of embodiments 14-24, wherein:

(a) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(b) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index).

26. The immunoconjugate of any one of embodiments 14-25, wherein the first subunit and/or the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index).

27. The immunoconjugate of any one of embodiments 1 -26, wherein the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1 .

28. The immunoconjugate of any one of embodiments 1 -26, wherein the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1 .

29. The immunoconjugate of any one of embodiments 1 -28, wherein binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and/or binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 .

30. The immunoconjugate of any one of embodiments 1 -28, wherein binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 or binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 .

31 . The immunoconjugate of any one of embodiments 1 -27, 29, and 30, wherein:

(a) binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 ; or

(b) binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 .

32. The immunoconjugate of any one of embodiments 1 -31 , wherein the DBA moiety comprises the following six complementarity-determining regions (CDRs):

(a) a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); or (b) a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75).

33. The immunoconjugate of embodiment 32, wherein the DBA moiety comprises:

(a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58;

(b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; or

(c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 ; or

(d) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 .

34. The immunoconjugate of embodiment 33, wherein the DBA moiety comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 76; or

(c) a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or

(d) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 91 .

35. The immunoconjugate of any one of embodiments 1 -34, wherein the anti-PD-1 antibody moiety comprises the following six CDRs:

(a) a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 );

(b) a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39); or

(c) a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR-H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3).

36. The immunoconjugate of embodiment 35, wherein the anti-PD-1 antibody moiety comprises:

(a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40; or

(c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

37. The immunoconjugate of embodiment 36, wherein the anti-PD-1 antibody moiety comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising the amino acid sequence of SEQ ID NO: 22;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising the amino acid sequence of SEQ ID NO: 40; or

(c) a VH comprising the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising the amino acid sequence of SEQ ID NO: 4.

38. The immunoconjugate of any one of embodiments 1 -31 , wherein:

(a) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); (b) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR- L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); or

(c) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR- L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39).

39. The immunoconjugate of embodiment 38, wherein:

(a) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(b) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(c) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; or

(d) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40.

40. The immunoconjugate of embodiment 39, wherein:

(a) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22;

(b) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22;

(c) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or

(d) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 40.

41 . The immunoconjugate of any one of embodiments 1 -40, wherein the linker is between 5 to 30 amino acids in length.

42. The immunoconjugate of embodiment 41 , wherein the linker is 20 amino acids in length.

43. The immunoconjugate of any one of embodiments 1 -41 , wherein the linker comprises the amino acid sequence (G2SG2)x, wherein x is an integer between 1 and 6 (SEQ ID NOs: 149-154).

44. The immunoconjugate of embodiment 43, wherein x is 2 (SEQ ID NO: 150) or 4 (SEQ ID NO: 152).

45. The immunoconjugate of any one of embodiments 1 -44, wherein the IL-2 polypeptide is connected to the VH of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]- C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus. 46. The immunoconjugate of any one of embodiments 1 -44, wherein the IL-2 polypeptide is connected to the VL of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus.

47. The immunoconjugate of any one of embodiments 1 -46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, and/or an alanine residue at position 125 (numbered relative to human IL-2 sequence of SEQ ID NO: 147).

48. The immunoconjugate of embodiment 47, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 146.

49. The immunoconjugate of any one of embodiments 1 -46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, an alanine residue at position 125, and/or a threonine residue at position 126 (numbered relative to human IL-2 sequence of SEQ ID NO: 147).

50. The immunoconjugate of embodiment 49, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 199.

51 . The immunoconjugate of any one of embodiments 1 -50, wherein the PD-1 is human PD-1 .

52. An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and

(iii) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor;

(b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein: (i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein:

(a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or

(b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule.

53. An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (Hi) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor; (b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein:

(i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein:

(a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or

(b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule.

54. An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and

(Hi) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor;

(b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is an scFv comprising a VH and a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein:

(i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

55. The immunoconjugate of any one of embodiments 1 -51 , wherein the immunoconjugate comprises:

(a) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(b) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(c) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(d) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128;

(e) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;

(f) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;

(g) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128;

(h) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; or (i) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.

56. The immunoconjugate of embodiment 55, wherein the immunoconjugate comprises:

(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(c) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(d) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128;

(e) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120;

(f) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120;

(g) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128;

(h) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; or (i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

57. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

58. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

59. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128.

60. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120.

61 . An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

62. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

63. An isolated polynucleotide or a set of isolated polynucleotides encoding the immunoconjugate of any one of embodiments 1 -62.

64. A vector or a set of vectors comprising the isolated polynucleotide or the set of isolated polynucleotides of embodiment 63.

65. A host cell or a set of host cells comprising (i) the isolated polynucleotide or the set of isolated polynucleotides of embodiment 63 or (ii) the vector or the set of vectors of embodiment 64.

66. A method of producing an immunoconjugate, comprising the steps of (a) culturing the host cell or the set of host cells of embodiment 65 under conditions suitable for the expression of the immunoconjugate.

67. The method of embodiment 66, further comprising recovering the immunoconjugate. 68. The method of embodiment 66, wherein the host cell expresses the first binding domain and the second binding domain.

69. The method of embodiment 66, wherein a first host cell expresses the first binding domain, and a second host cell expresses the second binding domain.

70. The method of embodiment 68 or 69, further comprising recovering the first binding domain and the second binding domain.

71 . The method of embodiment 70, further comprising contacting the recovered first binding domain with the recovered second binding domain.

72. An immunoconjugate produced by the method of any one of embodiments 66-71 .

73. A pharmaceutical composition comprising the immunoconjugate of any one of embodiments 1 -62 and 72 and a pharmaceutically acceptable carrier.

74. The immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73 for use as a medicament.

75. Use of the immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73 in the manufacture of a medicament.

76. The immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73 for use in the treatment of a cancer in a subject in need thereof.

77. Use of the immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73 in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof.

78. Use of the immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73 for treating a cancer in a subject in need thereof.

79. A method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of the immunoconjugate of any one of embodiments 1 -62 and 72 or the pharmaceutical composition of embodiment 73.

80. The immunoconjugate for use, pharmaceutical composition for use, use, or method of any one of embodiments 76-79, wherein the cancer is a PD-1 -positive cancer.

81 . The immunoconjugate for use, pharmaceutical composition for use, use, or method of any one of embodiments 76-79, further comprising administering an additional therapeutic agent to the subject.

LISTING OF SEQUENCES

Sequences disclosed herein are provided below in Table 29.

Table 29. Listing of Sequences Disclosed Herein.

OTHER EMBODIMENTS

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:

1 . An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide;

(ii) a linker; and

(iii) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus, and -C denotes a polypeptide C-terminus, and wherein the first binding domain is configured such that:

(i) when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor; and

(ii) when the DBA is bound to PD-1 , the DBA moiety is substantially blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to an IL-2 receptor; and

(b) a second binding domain comprising an anti-PD-1 antibody moiety comprising a VH and a VL.

2. The immunoconjugate of claim 1 , wherein the anti-PD-1 antibody moiety does not substantially bind to an IL-2 polypeptide.

3. The immunoconjugate of claim 1 or 2, wherein the DBA moiety comprises a Fab molecule.

4. The immunoconjugate of claim 3, wherein the DBA moiety comprises a Fab heavy chain comprising the VH of the DBA moiety and a heavy chain constant domain 1 (CH1 ) and a Fab light chain comprising the VL of the DBA moiety and a light chain constant domain (CL), wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

5. The immunoconjugate of claim 3, wherein the DBA moiety is a conventional Fab molecule.

6. The immunoconjugate of any one of claims 1 -5, wherein the anti-PD-1 antibody moiety comprises a Fab molecule.

7. The immunoconjugate of claim 6, wherein the anti-PD-1 antibody moiety comprises a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

8. The immunoconjugate of any one of claims 1 -5, wherein:

(a) the DBA moiety is a conventional Fab molecule; and

(b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other.

9. The immunoconjugate of any one of claims 1 -5, wherein:

(a) the DBA moiety is a conventional Fab molecule; and

(b) the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other.

10. The immunoconjugate of claim 6, wherein the anti-PD-1 antibody moiety is a conventional Fab molecule.

11 . The immunoconjugate of any one of claims 1 -5, wherein:

(a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other or the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and

(b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

12. The immunoconjugate of any one of claims 1 -5, wherein:

(a) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and

(b) the anti-PD-1 antibody moiety is a conventional Fab molecule.

13. The immunoconjugate of any one of claims 1 -5, wherein the anti-PD-1 antibody moiety is a single-chain variable fragment (scFv).

14. The immunoconjugate of any one of claims 1 -13, further comprising an Fc domain comprising a first subunit and a second subunit.

15. The immunoconjugate of any one of claims 1 -14, wherein (a) the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus; and/or (b) the anti-PD-1 antibody moiety and the second subunit are connected in the following orientation: N-[anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

16. The immunoconjugate of claim 14 or 15, wherein the Fc domain is an IgG Fc domain.

17. The immunoconjugate of claim 16, wherein the IgG Fc domain is an IgG 1 Fc domain.

18. The immunoconjugate of any one of claims 14-17, wherein the Fc domain is a human IgG Fc domain.

19. The immunoconjugate of any one of claims 14-18, wherein the first subunit comprises one or more CH domains selected from a first CH2 (CH2i) domain and/or a first CH3 (CH3i) domain; and the second subunit comprises one or more CH domains selected from a second CH2 (CH22) domain and/or a second CH3 (CH32) domain.

20. The immunoconjugate of claim 19, wherein at least one of the one or more CH domains is paired with another CH domain.

21 . The immunoconjugate of claim 20, wherein the CH3i and CH32 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH3i domain is positionable in the cavity or protuberance, respectively, in the CH32 domain.

22. The immunoconjugate of claim 21 , wherein the CH3i and CH32 domains meet at an interface between the protuberance and cavity.

23. The immunoconjugate of any one of claims 20-22, wherein the CH2i and CH22 domains each comprise a protuberance or cavity, and wherein the protuberance or cavity in the CH2i domain is positionable in the cavity or protuberance, respectively, in the CH22 domain.

24. The immunoconjugate of claim 23, wherein the CH2i and CH22 domains meet at an interface between the protuberance and cavity.

25. The immunoconjugate of any one of claims 14-24, wherein:

(a) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(b) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index).

26. The immunoconjugate of any one of claims 14-25, wherein the first subunit and/or the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index).

27. The immunoconjugate of any one of claims 1 -26, wherein the DBA moiety and the anti-PD-1 antibody moiety bind to different epitopes of PD-1 .

28. The immunoconjugate of any one of claims 1 -26, wherein the DBA moiety and the anti-PD-1 antibody moiety bind to the same epitope of PD-1 .

29. The immunoconjugate of any one of claims 1 -28, wherein binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and/or binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1.

30. The immunoconjugate of any one of claims 1 -28, wherein binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 or binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 .

31 . The immunoconjugate of any one of claims 1 -27, 29, and 30, wherein:

(a) binding of the DBA moiety to PD-1 inhibits binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 ; or

(b) binding of the DBA moiety to PD-1 does not inhibit binding of PD-1 to PD-L1 and binding of the anti-PD-1 antibody moiety to PD-1 inhibits binding of PD-1 to PD-L1 .

32. The immunoconjugate of any one of claims 1 -31 , wherein the DBA moiety comprises the following six complementarity-determining regions (CDRs):

(a) a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); or

(b) a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75).

33. The immunoconjugate of claim 32, wherein the DBA moiety comprises:

(a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58;

(b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; or

(c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 ; or

(d) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 91 .

34. The immunoconjugate of claim 33, wherein the DBA moiety comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 76; or

(c) a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 91 ; or

(d) a VH comprising the amino acid sequence of SEQ ID NO: 85 and a VL comprising the amino acid sequence of SEQ ID NO: 91 .

35. The immunoconjugate of any one of claims 1 -34, wherein the anti-PD-1 antibody moiety comprises the following six CDRs:

(a) a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 );

(b) a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR-L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39); or

(c) a CDR-H1 comprising the amino acid sequence of SYWMS (SEQ ID NO: 10), a CDR-H2 comprising the amino acid sequence of AISGSGGSRYYAESVKG (SEQ ID NO: 11 ), a CDR-H3 comprising the amino acid sequence of SPLQWIDV (SEQ ID NO: 12), a CDR-L1 comprising the amino acid sequence of RASQGISSWLA (SEQ ID NO: 1 ), a CDR-L2 comprising the amino acid sequence of EASSLQS (SEQ ID NO: 2), and a CDR-L3 comprising the amino acid sequence of QQANQFPFT (SEQ ID NO: 3).

36. The immunoconjugate of claim 35, wherein the anti-PD-1 antibody moiety comprises:

(a) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(b) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40; or

(c) a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4.

37. The immunoconjugate of claim 36, wherein the anti-PD-1 antibody moiety comprises:

(a) a VH comprising the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising the amino acid sequence of SEQ ID NO: 22;

(b) a VH comprising the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising the amino acid sequence of SEQ ID NO: 40; or

(c) a VH comprising the amino acid sequence of SEQ ID NO: 13 and/or a VL comprising the amino acid sequence of SEQ ID NO: 4.

38. The immunoconjugate of any one of claims 1 -31 , wherein:

(a) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR-L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 );

(b) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of AYYIH (SEQ ID NO: 82), a CDR-H2 comprising the amino acid sequence of WIHPYSGMTNYAQKFQG (SEQ ID NO: 83), a CDR-H3 comprising the amino acid sequence of GHYYGKTDY (SEQ ID NO: 84), a CDR-L1 comprising the amino acid sequence of RASQGIRNDLG (SEQ ID NO: 73), a CDR-L2 comprising the amino acid sequence of GASSLQS (SEQ ID NO: 74), and a CDR-L3 comprising the amino acid sequence of QESYTSSNT (SEQ ID NO: 75); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYTMS (SEQ ID NO: 28), a CDR-H2 comprising the amino acid sequence of AISGGGRDIYYPDSVKG (SEQ ID NO: 29), a CDR-H3 comprising the amino acid sequence of LTGRVYFALDS (SEQ ID NO: 30), a CDR-L1 comprising the amino acid sequence of KASESVDTSDNSFIH (SEQ ID NO: 19), a CDR- L2 comprising the amino acid sequence of RASTLES (SEQ ID NO: 20), and a CDR-L3 comprising the amino acid sequence of QQNYDVPWT (SEQ ID NO: 21 ); or

(c) (i) the DBA moiety comprises a CDR-H1 comprising the amino acid sequence of RYYVH (SEQ ID NO: 64), a CDR-H2 comprising the amino acid sequence of IINPSGGYASYAQKFQG (SEQ ID NO: 65), a CDR-H3 comprising the amino acid sequence of GLFI (SEQ ID NO: 66), a CDR-L1 comprising the amino acid sequence of RASQSIGRYLA (SEQ ID NO: 55), a CDR-L2 comprising the amino acid sequence of SASNLET (SEQ ID NO: 56), and a CDR-L3 comprising the amino acid sequence of QQYNSFPVT (SEQ ID NO: 57); and (ii) the anti-PD-1 antibody moiety comprises a CDR-H1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 46), a CDR-H2 comprising the amino acid sequence of VITGSGGSTYYADSVKG (SEQ ID NO: 47), a CDR-H3 comprising the amino acid sequence of GEGYAGSSYFRASDI (SEQ ID NO:48), a CDR- L1 comprising the amino acid sequence of RASQSISSYLN (SEQ ID NO: 37), a CDR-L2 comprising the amino acid sequence of TASSLQS (SEQ ID NO: 38), and a CDR-L3 comprising the amino acid sequence of QQSYSTPLT (SEQ ID NO: 39).

39. The immunoconjugate of claim 38, wherein:

(a) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(b) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 103 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

(c) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 111 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 31 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22; or

(d) (i) the DBA moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 67 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 49 and/or a VL comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 40.

40. The immunoconjugate of claim 39, wherein:

(a) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22;

(b) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 103 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22;

(c) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 109; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 31 and a VL comprising the amino acid sequence of SEQ ID NO: 22; or

(d) (i) the DBA moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 67 and a VL comprising the amino acid sequence of SEQ ID NO: 58; and (ii) the anti-PD-1 antibody moiety comprises a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 40.

41 . The immunoconjugate of any one of claims 1 -40, wherein the linker is between 5 to 30 amino acids in length.

42. The immunoconjugate of claim 41 , wherein the linker is 20 amino acids in length.

43. The immunoconjugate of any one of claims 1 -41 , wherein the linker comprises the amino acid sequence (G2SG2)x, wherein x is an integer between 1 and 6 (SEQ ID NOs: 149-154).

44. The immunoconjugate of claim 43, wherein x is 2 (SEQ ID NO: 150) or 4 (SEQ ID NO: 152).

45. The immunoconjugate of any one of claims 1 -44, wherein the IL-2 polypeptide is connected to the VH of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VH]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus.

46. The immunoconjugate of any one of claims 1 -44, wherein the IL-2 polypeptide is connected to the VL of the DBA moiety in the following orientation: N-[IL-2 polypeptide]-[linker]-[VL]-C, wherein N- denotes a polypeptide N-terminus, and -C denotes a polypeptide C-terminus.

47. The immunoconjugate of any one of claims 1 -46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, and/or an alanine residue at position 125 (numbered relative to human IL-2 sequence of SEQ ID NO: 147).

48. The immunoconjugate of claim 47, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 146.

49. The immunoconjugate of any one of claims 1 -46, wherein the IL-2 polypeptide is a mutant human IL-2 polypeptide comprising an alanine residue at position 3, an alanine residue at position 42, an alanine residue at position 45, a glycine residue at position 72, an alanine residue at position 125, and/or a threonine residue at position 126 (numbered relative to human IL-2 sequence of SEQ ID NO: 147).

50. The immunoconjugate of claim 49, wherein the mutant human IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 199.

51 . The immunoconjugate of any one of claims 1 -50, wherein the PD-1 is human PD-1 .

52. An immunoconjugate comprising: (a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and

(iii) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor;

(b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein:

(i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein:

(a) the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other; or

(b) the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the DBA moiety and a CH1 and a Fab light chain comprising the VL of the DBA moiety and a light chain CL, wherein the CH1 of the Fab heavy chain and CL of the Fab light chain are replaced by each other; and the anti-PD-1 antibody moiety is a conventional Fab molecule.

53. An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 199;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 152; and (Hi) a DBA moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor;

(b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein:

(i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the DBA moiety is a conventional Fab molecule; and the anti-PD-1 antibody moiety is a Fab molecule comprising a Fab heavy chain comprising the VH of the anti-PD-1 antibody moiety and a CH1 and a Fab light chain comprising the VL of the anti-PD-1 antibody moiety and a CL, wherein the VH of the Fab heavy chain and the VL of the Fab light chain are replaced by each other.

54. An immunoconjugate comprising:

(a) a first binding domain comprising:

(i) an IL-2 polypeptide comprising the amino acid sequence of SEQ ID NO: 146;

(ii) a linker comprising or consisting of the amino acid sequence of SEQ ID NO: 150 or 152; and

(Hi) a dual binding antibody (DBA) moiety that binds to PD-1 and the IL-2 polypeptide in a mutually exclusive manner, wherein the DBA moiety is a Fab molecule comprising a Fab heavy chain comprising a VH and a Fab light chain comprising a VL, wherein the IL-2 polypeptide, the linker, and the DBA moiety are connected in the following orientation: N-[IL-2 polypeptide]-[linker]-[DBA moiety]-C, wherein N- denotes a polypeptide N- terminus and -C denotes a polypeptide C-terminus, and wherein when the DBA moiety is bound to the IL-2 polypeptide, the IL-2 polypeptide is substantially blocked from binding to its receptor, and when the DBA moiety is bound to PD-1 , the DBA moiety is blocked from binding to the IL-2 polypeptide and the IL-2 polypeptide is capable of binding to its receptor;

(b) a second binding domain comprising an anti-PD-1 antibody moiety, wherein the anti-PD-1 antibody moiety is an scFv comprising a VH and a VL; and

(c) an Fc region comprising a first subunit and a second subunit, wherein:

(i) the first subunit comprises a tryptophan residue at position 366; and the second subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407 (numbered according to Kabat EU index); or

(ii) the first subunit comprises a serine residue at position 366, an alanine residue at position 368, and a valine residue at position 407; and the second subunit comprises a tryptophan residue at position 366 (numbered according to Kabat EU index), and wherein each of the first subunit and the second subunit comprises an alanine residue at position 234, an alanine residue at position 235, and a glycine residue at position 329 (numbering according to Kabat EU index), wherein the DBA moiety and the first subunit are connected in the following orientation: N-[DBA moiety]-[first subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus, and wherein the anti-PD-1 antibody moiety and the second subunit are connected according to N- [anti-PD-1 antibody moiety]-[second subunit]-C, wherein N- denotes a polypeptide N-terminus and -C denotes a polypeptide C-terminus.

55. The immunoconjugate of any one of claims 1 -51 , wherein the immunoconjugate comprises:

(a) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(b) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 10, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(c) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 12, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 10, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;

(d) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128;

(e) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 18, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;

(f) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 120;

(g) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 128;

(h) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108; or

(i) a first polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 108.

56. The immunoconjugate of claim 55, wherein the immunoconjugate comprises:

(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(b) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(c) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 112, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108;

(d) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128;

(e) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120;

(f) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120;

(g) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 122, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128;

(h) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108; or

(i) a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

57. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 96, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

58. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 104, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 110, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

59. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 126, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 128.

60. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 124, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 134, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 118, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 120.

61 . An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 197, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

62. An immunoconjugate comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 198, a second polypeptide comprising the amino acid sequence of SEQ ID NO: 94, a third polypeptide comprising the amino acid sequence of SEQ ID NO: 106, and a fourth polypeptide comprising the amino acid sequence of SEQ ID NO: 108.

63. An isolated polynucleotide or a set of isolated polynucleotides encoding the immunoconjugate of any one of claims 1 -62.

64. A vector or a set of vectors comprising the isolated polynucleotide or the set of isolated polynucleotides of claim 63.

65. A host cell or a set of host cells comprising (i) the isolated polynucleotide or the set of isolated polynucleotides of claim 63 or (ii) the vector or the set of vectors of claim 64.

66. A method of producing an immunoconjugate, comprising the steps of (a) culturing the host cell or the set of host cells of claim 65 under conditions suitable for the expression of the immunoconjugate.

67. The method of claim 66, further comprising recovering the immunoconjugate.

68. The method of claim 66, wherein the host cell expresses the first binding domain and the second binding domain.

69. The method of claim 66, wherein a first host cell expresses the first binding domain, and a second host cell expresses the second binding domain.

70. The method of claim 68 or 69, further comprising recovering the first binding domain and the second binding domain.

71 . The method of claim 70, further comprising contacting the recovered first binding domain with the recovered second binding domain.

72. An immunoconjugate produced by the method of any one of claims 66-71 .

73. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 -62 and 72 and a pharmaceutically acceptable carrier.

74. The immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73 for use as a medicament.

75. Use of the immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73 in the manufacture of a medicament.

76. The immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73 for use in the treatment of a cancer in a subject in need thereof.

77. Use of the immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73 in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof.

78. Use of the immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73 for treating a cancer in a subject in need thereof.

79. A method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of the immunoconjugate of any one of claims 1 -62 and 72 or the pharmaceutical composition of claim 73.

80. The immunoconjugate for use, pharmaceutical composition for use, use, or method of any one of claims 76-79, wherein the cancer is a PD-1 -positive cancer.

81 . The immunoconjugate for use, pharmaceutical composition for use, use, or method of any one of claims 76-79, further comprising administering an additional therapeutic agent to the subject.